Citrated Platelet Rich Plasma Research Articles

Effects of Blood-Derived Products on Cellular Senescence and Inflammatory Response: A Study on Skin Rejuvenation.

Blood-derived products, such as citrate platelet-rich plasma (CPRP) and hyperacute serum (HAS), are recognized for their rich growth factor content. When human dermal fibroblast (HDF) cells are exposed to combined mitogenic and DNA-damaging stimuli, it can lead to an increased burden of senescent cells and a modified senescence-associated secretory phenotype. In this study, the senescent state was comprehensively assessed through various methods, including phosphorylated histone H2AX (γH2AX) staining, p21 and p16 q-PCR, p21-western blot, growth curves, and senescence-associated ß-galactosidase staining. Two primary treatments with blood products were administered, one early (immediately after etoposide) and the other late (11 days after etoposide treatment). The effects of the blood product treatment were evaluated by measuring interleukin 6 and 8 (IL-6 and IL-8) levels, as well as collagen 1 (COL1) and p21 mRNA expression. Additionally, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assays, cell size measurements, viability assays, and cell number calculations were conducted. The results revealed that cells treated with hyperacute serum in the early treatment phase exhibited the lowest observed IL-6 and IL-8 levels. In contrast, a clear inflammatory response for IL-8 was observed in cells treated with hyperacute serum and citrate platelet-rich plasma during the late treatment. Furthermore, an upregulation of COL1 expression was observed in the early treatment, while cells in the late treatment group remained unaffected. Notably, citrate platelet-rich plasma-treated cells showed a decrease in COL1 expression. Overall, the treatment with blood products appears to have slightly positive effects on skin rejuvenation.

The Composition of Hyperacute Serum and Platelet-Rich Plasma Is Markedly Different despite the Similar Production Method.

Autologous blood derived products, such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are widely applied in regenerative therapies, in contrast to the drawbacks in their application, mainly deriving from the preparation methods used. Eliminating the disadvantages of both PRP and PRF, hyperacute serum (HAS) opens a new path in autologous serum therapy showing similar or even improved regenerative potential at the same time. Despite the frequent experimental and clinical use of PRP and HAS, their protein composition has not been examined thoroughly yet. Thus, we investigated and compared the composition of HAS, serum, PRP and plasma products using citrate and EDTA by simple laboratory tests, and we compared the composition of HAS, serum, EDTA PRP and plasma by Proteome Profiler and ELISA assays. According to our results the natural ionic balance was upset in both EDTA and citrate PRP as well as in plasma. EDTA PRP contained significantly higher level of growth factors and cytokines, especially platelet derived angiogenic and inflammatory proteins, that can be explained by the significantly higher number of platelets in EDTA PRP. The composition analysis of blood derivatives revealed that although the preparation method of PRP and HAS were similar, the ionic and protein composition of HAS could be advantageous for cell function.

Evaluation of reticulated platelets in dogs with breed-related thrombocytopenia.

The aim of this study was to evaluate the percentage of reticulated platelets in healthy dogs with breed-related thrombocytopenia. Seventy two dogs, clinically healthy, were enrolled in the study. Blood was collected from the patients and anticoagulated with tripotassium ethylenediaminetetraacetic acid (K3-EDTA) and sodium citrate. Platelet count was obtained by an impedance haematology analyser and platelet morphology was evaluated by examination of blood smears. Patients were allocated into two groups. Group 1 consisted of 30 dogs with normal platelet count, whereas group 2 was composed of 42 dogs with thrombocytopenia. Thrombocytopenia was present in both K3-EDTA and citrate blood samples. Patients with thrombocytopenia were divided into two subgroups: the first subgroup included dogs with platelet count in K3-EDTA anticoagulated blood from 100 to 200 x10(9)/L, patients in the second subgroup had a platelet count of less than 100 x10(9)/L. The percentage of young reticulated platelets (RPs) labelled with thiazole orange, and the percentage of platelets coated with platelet surface-associated IgG, were determined in platelet-rich plasma (PRP) by a flow cytometer. The mean percentage of RPs in K3-EDTA and citrate PRP was significantly higher in dogs with thrombocytopenia than in dogs with normal platelet count. The mean percentage of RPs was significantly higher in citrate PRP than in K3-EDTA PRP in all groups. The results suggest that idiopathic, asymptomatic thrombocytopenia is not caused by platelet surface-associated IgG. Dogs with breed-related thrombocytopenia have a competent bone marrow.

A Synthetic Evaluation Of Genetic and Pharmacological Resistance To Clopidogrel and On Treatment Residual Platelet Aggregation In Patients With Atherothrombosis

BackgroundClopidogrel is cornerstone treatment for atherothrombotic patients. The incidence of recurrent thrombotic events raises up to 10% of patients on treatment with clopidogrel. Intestinal absorption via the glycoproteine ABCB1, bioactivation by the cytochrome P450 (CYP) system, binding to P2Y12 receptor and inhibition of platelet aggregation are critical steps for the efficiency of clopidogrel treatment. Prospective independent clinical trials showed that the risk of recurrent thrombotic complications is associated with the presence of common polymorphisms of genes encoding ABCB1 (rs1045642C>T) or the CYP2C19 isoenzyme (*2_rs4244285G>A, *3_rs4986893G>A, *4_rs28399504A>G) or the inefficiency of clopidogrel to decrease intracellular VASP or the presence of high residual platelet reactivity (HRPR). AimTo evaluate the available pharmacogenetic, pharmacological and functional assays for the diagnosis of the resistance to clopidogrel treatment on the same population of patients with atherothrombosis. Materials and MethodsA cohort of 94 out-patients with atherothrombosis receiving clopidogrel treatment (75 mg o.d) for more than 3 months were included. Polymorphisms ABCB1, CYP *2, *3, *4, *17 were assessed with rPCR method. VASP was measured with flow cytometry assay and poor response (PR) to the treatment was diagnosed if VASP index was > 50. Light Transmission Platelet Aggregation (LTPA) triggered by ADP (5 ìM and 10 ìM) was assessed on citrated platelet rich plasma. Multielectrode aggregometry (WBMEA) was performed on citrated whole blood using Multiplate instrument and ADP reagent (ADP-test®). The cut-off point for the diagnosis of HRPR on clopidogrel treatment was the lower value of the maximum aggregation or the AUC observed in the upper quintile of ADP triggered LTPA or WBMEA measurements. ResultsThe frequency of heterozygous for CYP*2, *3, *4 and ABCB 1 polymorphisms was 28%, 0%, 1%, and 51% respectively. The frequency of the respective homozygocity was 2%, 0%, 0%, and 20%. Patients heterozygous for the CYP*2 G>A polymorphism showed significantly higher VASP index as compared to the wild type but LTPA and WBMEA were not significantly different. VASP index, LTPA and WBMEA were not significantly different in patients heterozygous or homozygous for the ABCB1 C>T polymorphism as compared to the wild type. The VASP index was significantly correlated with the LTPA (r=0.44, p<0.0001) and WBMEA (r=0.376, p=0.001). In addition LTPA was significantly correlated with the WBMEA (r=0.46, p<0.0001). With the VASP assay 42% of patients were diagnosed as PR to clopidogrel. HRPR was found in 12% and 28% of patients tested with LTPA ADP 5 ìM and 10 ìM respectively and in 19% of patients tested with WBMEA. Among patients with HRPR the frequency of PR with the VASP assay was 60% when they were tested with LTPA ADP 10 ìM, 70% when they were tested with LTPA ADP 5 ìM and 76% when they were tested with WBMEA. Less than 50% of patients with HRPR had at least one of the studied polymorphisms. One patient homozygous for CYP*2 and ABCB1 was PR with the VASP assay and had HRPR with the WBMEA.CYP *2G>ACYP*17 C>TABCB1 C>TWild typeHeterozygousWild typeHeterozygousHomozygousWild typeHeterozygousHomozygousVASP index42±1854±20*48±2040±1838±951±2043±1744±23LTA ADP 10 ìM47±2048±2150±2045±2033±6*51±2245±2149±15LTA ADP 5 ìM36±1737±1939±1834±1827±6*38±2235±1739±11WB-MEA ADP261±113333±173*294±113292±176176±97*302±176280±114272±138 ConclusionThe present study performed on a cohort of patients with atherothrombosis on treatment with clopidogrel demonstrates that the genotype of CYP and ABCB1 polymorphisms have limited influence on the pharmacological response to the treatment and on the residual platelet reactivity. Among the studied polymorphisms only the CYP*2G>A was related with increase of VASP index and WBMEA. Pharmacological response to clopidogrel is correlated with the level of residual platelet reactivity. However 25-30% of patients with HRPR showed good pharmacological response to clopidogrel. The present study underlines the need to a synthetic use of bioassays for the evaluation of the risk of failure of clopidogrel treatment to prevent the recurrence of atherothrombosis. The need for the construction of a diagnostic algorithm including the genetic, pharmacological and functional assays is revealed by the present study. Disclosures:No relevant conflicts of interest to declare.

A Locally Administered Novel Heparin Complex Attenuates Collagen-Induced Platelet Activation and Thrombosis In Vivo,

Abstract 3361We have developed a semi-synthetic antithrombotic heparin complex, APL001, to mimic mast cell-derived natural heparin proteoglycans (HepPG). HepPG attenuate platelet-collagen interactions under blood flow by inhibiting VWF- and GPIIb/IIIa -mediated platelet aggregation. In addition, rat-derived HepPG arrest platelet thrombus growth on collagen surfaces or at vascular injury sites, both in vitro and in vivo (Lassila et al.ATVB 1997, Kauhanen et al. ATVB 2000, Olsson et al. Thromb Haemost 2002).Our objective was to study the inhibitory capacity of APL001 for preventing human platelet aggregation in vitro and acute thrombosis in a baboon model in vivo. The effects of unfractionated heparin (UFH) and APL001 were compared in relevant coagulation assays (APTT, PT, thrombin time, anti-FXa activity, fibrinogen, FVIII:C and VWF activity (VWF:RCo) and antigen). Additionally, agonist-induced (collagen, ristocetin and ADP) platelet aggregation in citrate or hirudin-anticoagulated whole blood (Multiplate®) (n=10 healthy subjects), and platelet function analysis (PFA100®) in citrated platelet rich plasma (PRP) were assessed. In a well-established baboon thrombosis model a collagen-coated PTFE graft (length 2 cm, lumen 4 mm) was placed in an arterio-venous shunt. Prior to blood contact the thrombogenic surface was treated for 10 min with UFH or APL001 (both at 4 mg/mL). Thrombus formation was initiated by exposing the surface to blood flow (100 mL/min, shear rate 265−1), and the deposition of 111-In-labeled platelets and of fibrin was quantified continuously over 1h. Fibrin thrombus accumulation was assessed from the incorporation of circulating 125-I-fibrinogen.In the heparin-relevant coagulation tests APL001 was comparable or 20–30% more potent than UFH while FVIII, fibrinogen and VWF variables remained unaltered. In contrast to UFH, APL001 (300 μg/mL) consistently inhibited collagen- and ristocetin-induced platelet aggregation, whereas UFH had only a modest effect in comparison with PBS control (Table). ADP-induced aggregation was unaffected. Comparable results were observed in the PRP aggregation assay. PFA100 testing also demonstrated inhibitory effects. In the in vivo thrombosis model (n=4) APL001 reduced platelet deposition on collagen (vs. the results with UFH) by 34% (p=0.01), while platelet accumulation in distal propagated thrombus was reduced by 61% (p=0.16). APL001-treated surfaces accumulated 45% less fibrin than the UFH-treated surfaces (p=0.008).In conclusion, when compared with UFH APL001 inhibited both collagen- and ristocetin-induced platelet aggregation in human blood, while anticoagulant properties were comparable. In the absence of systemic antithrombotic drugs, exposure of APL001 to a highly thrombogenic collagen surface arrested thrombus formation in an in vivo baboon model. This finding suggests that locally administered APL001 alone, due to its dual antiplatelet and anticoagulant effects, may limit the growth and size of thrombus and thereby prevent subsequent thrombo-occlusion.TableAnticoagulantInhibition-% of platelet aggregation ± SDConc. 300 μg/mLnColl (3.2 μg/mL)Ristocetin (0.77 mg/mL)ADP (6.4 μM)CitrateAPL0011033 ± 1543 ± 166 ± 24UFH1011 ± 1323 ± 153 ± 7p value0.0030.0100.700HirudinAPL0011032 ± 1043 ± 178 ± 10UFH108 ± 1116 ± 166 ± 9p value0.0000.0020.600 Disclosures:Lassila:Aplagon: Chief Scientific Advisor.

Historical perspective on ADP-induced platelet activation

We have come a long way since 1960 when Hellem observed that a small molecule lost from red cells caused platelets to adhere to glass [1]. During the next year, Ollgaard [2] showed that this small molecule caused platelet aggregation, and it was identified as adenosine diphosphate (ADP) in Owren’s laboratory [3]. At that time, Marjory Zucker was grading ADP-induced aggregation visually as 1–4 after shaking citrated platelet-rich plasma by hand for 1 min [4]. She had been carrying out research on platelets since 1947 and was already a major figure in the field. The key development of the aggregometer in Gustav Born’s laboratory in 1962 provided a light transmission technique for assessing and recording the rate and extent of aggregation that is still widely used today (light transmission aggregometry [LTA]) [5]. In the next few years, Born and his colleagues used the aggregometer in detailed investigations of the changes in platelets during ADP-induced aggregation, and inhibitors of this process [6]. Much of the early work was well reviewed in the classic book The Physiology of Blood Platelets, subtitled Recent Biochemical, Morphologic and Clinical Research, that was written by Aaron Marcus and Marjorie Zucker in 1965 [7]. In 1970, another major review summarized the developments in the 1960s [6]. It had become obvious that ADP plays an important role in hemostasis and thrombosis. The much more recent findings that platelets possess two P2Y receptors (P2Y1 and P2Y12) for ADP, and a P2X1 receptor for ATP have made it possible to understand the reactions responsible for many of the early observations [8]. Our present knowledge about ADP-induced platelet activation is attributable to the work of thousands of investigators and this historical review can mention only some of them. In the late 1950s and early 1960s, several groups of investigators carried out in vitro experiments showing that thrombin or collagen caused platelet aggregation and that ADP was released during this process [9–13]. In vivo, ADP and ATP do not normally circulate in the plasma, but they are stored in the dense granules of the platelets. During the formation of hemostatic plugs or arterial thrombi, platelets are stimulated by collagen and thrombin to release the contents of these platelet storage granules. In vitro, at a normal platelet count of 250,000/μl, the concentrations of ATP and ADP in plasma immediately after release of granule contents induced by collagen or thrombin have been reported in the ranges of 4–7 μM for ATP and 3–4 μM for ADP [14, 15]. The released ADP adds to the response of platelets to the other aggregating agents. In addition to causing aggregation, the effects of ADP on platelets include shape change, refractoriness, potentiation of the effects of other aggregating agents, inhibition of platelet adenylyl cyclase, increase in cytosolic free calcium, and activation of specific receptors that stimulate intracellular signaling pathways that converge on the cytoplasmic domain of the integrin αIIbβ3 (glycoprotein (GP) IIb–IIIa), leading to its becoming able to bind extracellular fibrinogen and von Willebrand factor [16, 17].

Activation of Caspases-8 and -9 Occurs In Platelets From Children with Immune, but Not Chemotherapy-Related, Thrombocytopenia.

AbstractAbstract 3700Apoptotic-like processes in platelets are similar to those observed during apoptosis in the cytoplasm of nucleated cells: activation of caspase-8, caspase-9, and caspase-3, loss of mitochondrial inner membrane potential, and externalization of phosphatidylserine (PS) (Leytin et al, Br J Haematol 2006; Lopez et al, J Thromb Haemost 2009). We recently showed that platelets in pediatric primary immune thrombocytopenia (ITP) have activated caspase-3 (aCASP3) and externalized PS, both of which were reduced after IVIg administration (Speer et al, Blood 2008;112: 3417). To gain a more complete understanding of the apoptosis that occurs in ITP platelets, in the present study, we investigated whether caspase-8 and caspase-9 are also activated in platelets from children with ITP, and examined whether the increase in platelet count in response to IVIg is associated with a decrease in activated caspase-8 (aCASP8) and -9 (aCASP9) in platelets, as was observed for aCASP3. In addition, we measured the mitochondrial membrane potential in platelets before and after IVIg therapy. Children with primary ITP were enrolled in this prospective study. Severity of bleeding symptoms was assessed according to a pediatric bleeding score for ITP at the time of diagnosis. Blood samples were obtained at the time of diagnosis and after IVIg therapy for measurement of platelet count and for flow cytometric analyses of platelet apoptotic-like events. In citrated platelet-rich plasma, platelets were identified as CD42 positive events; aCASP8 and aCASP9 were measured as % platelets with bound FITC-fluorescent-labeled inhibitors of activated caspases; and mitochondrial membrane potential was measured as mean fluorescence intensity of the membrane potential sensitive fluorescent tetramethylrhodamine ethyl ester (TMRE). All patients (median age 5.4 yrs, n = 8) presented with typical symptoms of acute ITP with a bleeding score of 2 – 3 and had platelet counts < 20×109/L. Results from ITP patients were compared with 2 control groups, healthy children (platelet counts: 266–348 × 109/L, median age 6.8 yrs, n = 7) and children with thrombocytopenia as a result of chemotherapy for malignancies (cTP) (platelet counts: 3–51 × 109/L, median age 10.2 yrs, n = 7). ITP patients had significantly higher proportions of platelets with aCASP8 (17.5±5.1%) and aCASP9 (16.9±5.8%) compared with both healthy children (aCASP8 1.0±0.3%; aCASP9 1.1±0.3%) and children with cTP, (aCASP8 2.2±0.4%; aCASP9 1.9±0.4%) (p<0.01-0.05). In contrast, a loss of mitochondrial membrane potential was not observed in platelets from ITP patients at baseline, in healthy controls, or cTP. All ITP patients were treated with a maximum of 3 doses of IVIg (0.4 – 0.8 g/kg/dose) and showed a rise in platelet counts to > 20 × 109/L and amelioration of bleeding symptoms by 24 – 72 hours after IVIg administration. Concomitantly, the fractions of platelets with aCASP8 and aCASP9, decreased towards control values (ITP patients after IVIg: aCASP8 7.8±5.3%; aCASP9 6.9±2.1; p=0.5 for both compared to controls). Again no change in mitochondrial potential was observed after IVIg. In summary, we have demonstrated enhancement of the platelet apoptotic-like processes of aCASP8 and aCASP9 specifically in pediatric primary ITP, which were not observed in cTP. However, the platelet mitochondrial membrane potential was unchanged in ITP (before and after IVIg) and did not differ compared cTP and healthy children. Consistent with primary ITP, the patients' platelet counts were low and increased with IVIg administration. In parallel, IVIg led to a decrease of aCASP8 and aCASP9 in the patients' platelets. Together with our previously reported results (Speer et al, Blood 2008;112: 3417), we show that apoptotic events in platelets such as activation of caspases-8, -9, and -3 and PS exposure are increased specifically in ITP but not in cTP, and are decreased after IVIg treatment. As we detected no loss of the mitochondrial membrane potential in platelets from ITP patients, it may be that apoptotic processes in these platelets are not activated by mitochondrial signaling, but rather via an extrinsic signaling cascade including caspase-8, leading to the activation of caspase-3 and caspase-9. However, the complete signaling pathway leading to caspase-8 activation in platelets of pediatric ITP remains to be elucidated. Disclosures:No relevant conflicts of interest to declare.

IVIG Ameliorates Thrombin Activation of Platelets and Endogenous Thrombin Potential In Pediatric Patients with ITP.

AbstractAbstract 3706Immune thrombocytopenia (ITP) is a common hematological disorder in children that can lead to severe bleeding symptoms. Previous studies in ITP patients have found autoantibodies that bind to glycoprotein (GP) complex GPIIb/IIIa or GPIb/IX (Kiefel V et al. Br J Hematol 1991) and thus can alter platelet function (Olsson A et al. Thromb Res 2002; Nissner H et al. Blood 1986). Although platelet function has been studied in chronic ITP and in pediatric ITP (Panzer S et al. Europ J Haemat 2007; Semple et al. Blood 1996), so far platelet function was not studied in response to IVIg treatment, which leads to an increase of platelet counts and reduced bleeding symptoms. Next to platelet count no other biological markers have been correlated to bleeding symptoms. Therefore we studied the effect of IVIg treatment on platelet function and endogenous thrombin potential (ETP) in children with newly diagnosed primary ITP. Bleeding symptoms were assessed according to a pediatric bleeding score for ITP at the time of diagnosis and venous blood samples were obtained at the time of diagnosis and after IVIg therapy for measurement of platelet count and for flow cytometric analyses of platelets. In citrated platelet-rich plasma platelets were identified as CD42 positive events; CD62p, CD63 and PAC-1 binding were measured as % platelets with bound fluorescence before and after thrombin stimulation. All patients (median age 6.5 yrs, n=18) presented with typical symptoms of acute ITP with bleeding scores of 2 – 3 and had platelet counts < 20×109/L. (fig. 1). Results from ITP patients were compared to healthy children (median age 6.8 yrs, n = 18) with normal platelet counts and to children with thrombocytopenia as a result of chemotherapy for malignancies (cTP; median age 10.2 yrs, n = 9; platelet counts of 3 – 51×109/L). Results were expressed as the % of platelets expressing the antigen and were analyzed by ANOVA and Wilcoxon test. At initial presentation platelets of ITP patients showed an increased surface expression of CD62p (mean ± SEM: 14.4 ± 3.7 %, n = 17; p<0.05) and CD63 (27.21 ± 5.35 %, n = 17; p<0.05) compared to platelets from cTP patients (3.7 ± 1.1; 9.29 ± 1.7 %, n = 9) and healthy controls (4.9 ± 1.4; 9.5 ± 2.2 %, n = 9). PAC-1 binding was not increased in any of these groups. After thrombin stimulation, platelet surface expression of CD62p, CD63 and PAC1 increased significantly (p<0.0001). Among the groups, thrombin-stimulated expression of CD62p, CD63 and PAC1 was reduced in ITP (55.75 ± 7.77%; 57.07 ± 7.97%; 30.73 ± 7.71%; p<0.01) and in cTP (51.76 ± 7.59%; 55.22 ± 10.71%; 15.51 ± 5.05%; p<0.001) compared to healthy children (84.36 ± 4%; 91.94 ± 1.82%; 69.15 ± 6.91%). ETP was reduced in both, ITP (179.2 ± 52.3 nM thrombin; p<0.01) and cTP (185.0 ± 101.6 nM; p<0.05) compared to healthy children (353.4 ± 33.3 nM). After IVIg treatment all ITP patients showed a rise in platelet counts to >= 20 × 109/L (mean 46 × 109/L, range 20 – 123 × 109/L) and amelioration of bleeding symptoms. Concomitantly thrombin-stimulated platelet surface expression of CD62p, CD63 and PAC1 (84.51 ± 5.04%; 85.32 ± 5.79%; 56.8 ± 7.88%; p range 0.3 – 0.6) and ETP (316.6 ± 48.3 nM; p=0.3) improved and were not different from results in healthy children. In summary we demonstrated that platelets of children with primary ITP at baseline show an increased CD62p surface expression while thrombin-stimulated platelet activation of ITP and cTP patients is decreased compared to healthy children. After IVIg thrombin-stimulated platelet activation and ETP is similar to that seen in healthy children, even before normalization of the platelet counts. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Thrombin Generation Assessment in Platelet Rich Plasma Offers Additional Criteria for Low Molecular Weight Heparin Antithrombotic Fingerprint Characterization.

Abstract 4177 IntroductionNational and international organizations recognize that low molecular weight heparins (LMWHs) are distinct entities and that they should not be used interchangeably in clinical practice. Physicochemical properties, such as molecular weight anti-Xa/anti-IIa ratios, are used by health authorities to establish LMWHs differentiation. LMWHs are multitargeted drugs in which small differences in physicochemical properties may lead to significantly different inhibition of blood coagulation. In the present in vitro study we have investigated if thrombin generation assay, reflecting the overall coagulation process, is a suitable tool for the LMWH variability evalution. Materials and methodsLMWHs (bemiparin, enoxaparin, nadroparin, dalteparin and tinzaparin) and unfractionated heparin (UFH) were added in vitro in normal citrated platelet rich plasma from 10 healthy individuals, at clinically relevant concentrations (ranging from 0.2 to 1 anti-Xa IU/ml). Thrombin generation was studied with Thrombogram-Thrombinoscope® assay using diluted thromboplastin (Innovin®, 1/1000 final dilution, Siemens France). The concentrations doubling the lag-time, the time to Peak, the IC50 for Peak, the endogenous thrombin potential (ETP) and the mean rate index of propagation phase (MRI) were determined for each compound. ResultsLMWHs compared on their anti-Xa activity basis showed variable inhibitory effect on thrombin generation. At equivalent anti-Xa concentrations tinzaparin was significantly more potent than the other LMWHs, being almost similar to UFH profile. Enoxaparin, nadroparin and dalteparin showed a similar inhibitory activity. Bemiparin had the lesser pronounced inhibitory effect on thrombin generation. The impact of anti-Xa and anti-IIa activities on each phase of thrombin generation process is different. Thrombogram chronometric parameters are mainly influenced by the anti-IIa activity. Similarly, ETP inhibition depends more on anti-IIa rather than anti-Xa activity. The MRI of the propagation phase is more sensitive to the anti-Xa activity of LMWHs. ConclusionThrombin generation assessment in platelet rich plasma allows to evaluate the anticoagulant fingerprint of LMWHs and to differentiate them on global and functional criteria. Each LMWH has a particular inhibitory impact on each phase of thrombin generation process. These functional criteria need to be standardized and probably required for a better characterization of LMWHs heterogeneity by health authorities. They could be used also to evaluate bioequivalence of generic and original LMWHs. Disclosures:No relevant conflicts of interest to declare.

Human articular chondrocytes on macroporous gelatin microcarriers form structurally stable constructs with blood-derived biological gluesin vitro

Biodegradable macroporous gelatin microcarriers fixed with blood-derived biodegradable glue are proposed as a delivery system for human autologous chondrocytes. Cell-seeded microcarriers were embedded in four biological glues-recalcified citrated whole blood, recalcified citrated plasma with or without platelets, and a commercially available fibrin glue-and cultured in an in vitro model under static conditions for 16 weeks. No differences could be verified between the commercial fibrin glue and the blood-derived alternatives. Five further experiments were conducted with recalcified citrated platelet-rich plasma alone as microcarrier sealant, using two different in vitro culture models and chondrocytes from three additional donors. The microcarriers supported chondrocyte adhesion and expansion as well as extracellular matrix (ECM) synthesis. Matrix formation occurred predominantly at sample surfaces under the static conditions. The presence of microcarriers proved essential for the glues to support the structural takeover of ECM proteins produced by the embedded chondrocytes, as exclusion of the microcarriers resulted in unstable structures that dissolved before matrix formation could occur. Immunohistochemical analysis revealed the presence of SOX-9- and S-100-positive chondrocytes as well as the production of aggrecan and collagen type I, but not of the cartilage-specific collagen type II. These results imply that blood-derived glues are indeed potentially applicable for encapsulation of chondrocyte-seeded microcarriers. However, the static in vitro models used in this study proved incapable of supporting cartilage formation throughout the engineered constructs.

Effects of anticoagulant on pH, ionized calcium concentration, and agonist-induced platelet aggregation in canine platelet-rich plasma

OBJECTIVE-To compare effects of 3.8% sodium citrate and anticoagulant citrate dextrose solution National Institutes of Health formula A (ACD-A) on pH, extracellular ionized calcium (iCa) concentration, and platelet aggregation in canine platelet-rich plasma (PRP). SAMPLE POPULATION-Samples from 12 dogs. PROCEDURES-Blood samples were collected into 3.8% sodium citrate (dilution, 1:9) and ACD-A (dilution, 1:5). Platelet function, pH, and iCa concentration were evaluated in PRP. Platelet agonists were ADP, gamma-thrombin, and convulxin; final concentrations of each were 20microm, 100nM, and 20nM, respectively. Washed platelets were used to evaluate effects of varying the pH and iCa concentration. RESULTS-Mean pH and iCa concentration were significantly greater in 3.8% sodium citrate PRP than ACD-A PRP. Platelet aggregation induced by ADP and gamma-thrombin was markedly diminished in ACD-A PRP, compared with results for 3.8% sodium citrate PRP. Anticoagulant had no effect on amplitude of convulxin-induced platelet aggregation. In washed platelet suspensions (pH, 7.4), there were no differences in amplitude of platelet aggregation induced by convulxin or gamma-thrombin at various iCa concentrations. Varying the pH had no effect on amplitude of aggregation induced by convulxin or gamma-thrombin, but the aggregation rate increased with increasing pH for both agonists. CONCLUSIONS AND CLINICAL RELEVANCE-Aggregation of canine platelets induced by ADP and gamma-thrombin was negligible in ACD-A PRP, which suggested an increase in extraplatelet hydrogen ion concentration inhibits signaling triggered by these agonists but not by convulxin. Choice of anticoagulant may influence results of in vitro evaluation of platelet function, which can lead to erroneous conclusions.

Von Willebrand Factor-Glycoprotein Ib-Alpha Interactions Play a Role in Generating Platelet Microparticles: Data from Ex Vivo and in Vitro Studies

Microparticles (MP) are circulating submicroscopic cell fragments expressing procoagulant phospholipids and haemostatic proteins such as tissue factor. Several evidences support a pro-haemostatic role of MP and suggest that the axis VWF-platelet GpIb might be critical in their generation. The aim of this study was to measure the levels of platelet-derived MP (PMP) and tissue factor-expressing MP (TFMP) in relationship with VWF levels and multimeric composition as well as shear stress exploiting ex vivo and in vitro natural models. PMP and TFMP were analyzed by flow cytometry and defined as Annexin V and GpIIb/IIIa or Tissue Factor positive events falling in a gate defined by 1 μm beads. MP were enumerated adding a known number of 7 μm beads to each test tube. In the first set of experiments, levels of PMP and TFMP were measured before and after DDAVP administration in 9 patients with mild hemophilia A. FVIII:C, VWF:Ag, PMP and TFMP were measured in plasma at baseline and 30 min, 1, 2, 4, 8 and 24 h after DDAVP 0.3 μg/kg, subcutaneously. A significant increase in the levels of PMP and TFMP (p<0.05 at all time points vs baseline) was observed after DDAVP; peak levels were achieved after 2–8 h. Correlation was found between the increase of VWF:Ag and FVIII:C levels and the peak levels of MP (r=0.62 and r=0.7, respectively). We then studied 27 patients with different types of von Willebrand disease (VWD) and 15 normal controls: citrated platelet rich plasma was exposed to increasing shear stress (from 0 to 90 dyne) for 45 seconds in a cone and plate viscometer and generated PMP were then enumerated by flow cytometry. In normal controls the number of MP at 25 dyne was significantly higher than the number at 0 dyne (p<0.05). In patients with type 1 VWD and type 2B PMP significantly increased as in normal controls (p<0.05 at 25 dyne), while in type 3 patients no PMP increase was observed (p n.s. till 90 dyne). Interestingly, in type 2M patients PMP started to increased significantly at 50 dyne (p<0.05), while type 2A patients had the same behaviour of type 3 patients. This study support the hypothesis that ultralarge VWF multimers released by the endothelium interact with platelets and trigger MP generation, both in vivo and in vitro.

Doubly stochastic Poisson distribution of platelet adhesion on material surfaces and its implication on fluorescence image analysis

An image based assay has been developed to quantify platelet adhesion on material surfaces. Briefly, citrated platelet rich plasma (PRP) is incubated with materials for 2 h to allow platelet adhesion on the surface, followed by fluorescence labeling of platelets with Celltracker Green. Multiple images are acquired by an automatic fluorescence microscope, IN Cell Analyzer 1000. Platelets are identified and counted by an automatic image analysis algorithm. We have observed that the variance of the counts is considerably greater than expected from simple distribution laws. Statistical analysis of that difference shows that these measurements will often follow a doubly stochastic Poisson process in which the variance is inherently very large. To overcome this, multiple images (n > or = 8 images/well, about 3% of total area) are necessary to achieve accurate counting. This method has been compared to the commonly used enzyme based platelet adhesion assay, lactate dehydrogenase (LDH) assay. It is concluded that the present method is only effective in quantifying adherent platelets when a large number of samples are used. However, this method does provide additional information on platelet morphology and spatial distribution, which is lacking in the LDH assay.

Is chronic fatigue syndrome associated with platelet activation?

Chronic fatigue syndrome (CFS) is a debilitating condition that has no known aetiology or pathophysiology. Recent investigations by other workers have suggested that individuals with CFS may have a hypercoagulable state. This study investigated various aspects of platelet activation and function in 17 patients with CFS and in 16 age-matched and sex-matched healthy controls. Platelet aggregation, platelet volume and coagulation tests were performed. Platelet aggregation was investigated by means of the photometric changes using citrated platelet-rich plasma, whole blood aggregation was calculated as the percentage fall in single platelet counts and the coagulation tests were performed on an automatic microcentrifugal analyser.A trend was observed for the patients to have lower aggregation results and a reduced mean platelet volume. However, this only reached statistical significance for one result; the rate of the aggregation slope by 1.0 microg/ml collagen [CFS patients, 18 (9-28) versus controls, 32.5 (19-36); Mann-Whitney U test, P = 0.029]. No significant differences were found for any of the measurements of coagulation. These results are in contrast to previously reported findings. However, due to the heterogeneous nature of the disease, and the resulting lifestyles of the patients, caution should be taken when comparing one group of patients with another. Nevertheless, we certainly found no evidence of increased platelet activation or of a hypercoagulable state in patients with CFS and, on the basis of these results, anti-platelet or anti-coagulant therapy is not warranted.

The death of human platelets during incubation in citrated plasma involves shedding of CD42b and aggregation of dead platelets

The ability to readily identify dead platelets is invaluable to studies examining the means of their death, factors affecting their lifespan and their means of clearance by phagocytes. The aim of the present work was to develop a vital staining procedure for the rapid and objective discrimination of live from dead platelets that accrued in citrated platelet rich plasma (cPRP) incubated at 37 degrees C for several days. By transmission electron microscopy it was noted that platelet death was morphologically similar to necrosis and associated with aggregate formation. The vital dyes calcein-AM and FM 4-64 were found to robustly report the death of platelets and indicated that the aggregates which formed during incubation were populated exclusively by dead platelets. Additionally, platelet death was associated with the shedding of CD42b. Microscopic and cytometric analyses of incubated cPRP indicated that shedding of CD42b and aggregate formation by dead platelets were completely inhibited by the metalloproteinase inhibitor GM6001. Automated counting of platelets incubated in the presence of GM6001 revealed that death did not lead to a loss in cellularity. It is proposed that calcein-AM and FM4-64 are effective as vital stains for the reliable assessment of platelet viability and that platelet aggregation can occur by a novel mechanism dependent upon platelet death and metalloproteinase activity.

Influence of Stent Length and Heparin Coating on Platelet Activation: A Flow Cytometric Analysis in a Pulsed Floating Model

Platelets are involved in acute and subacute thrombotic occlusions of coronary stents and also may play a role in the pathophysiology of in-stent restenosis. This study sought to investigate the expression of activation dependent glycoproteins on platelets by flow cytometry and time until stent thrombosis in an in vitro model of stent thrombosis. Coronary stents were placed in parallel silicon tubings with circulating citrated platelet rich plasma to measure 1) influence of stent length on platelet antigens; 2) influence of heparin coating on platelet antigens; and 3) time until stent thrombosis. After recalcification aliquots of platelet-rich plasma were taken over 10 minutes in 2-minute intervals and immediately fixed and stabilized. For flow cytometric analysis monoclonal antibodies to CD41a (glycoprotein IIb/IIIa), CD42b (glycoprotein Ib-V-IX), CD62p (P-selectin), and CD63 (glycoprotein 53) were used. Within 2 minutes after start of circulation, the expression of CD62p and CD63 increased. Longer stents resulted in more platelet activation than shorter stents (25 mm vs. 15 mm; p<0.001. Time until stent thrombosis was reduced (25 mm vs. 15 mm; p<0.05). Heparin coating did not significantly influence flow cytometry detectable platelet activation but prolonged time until stent thrombosis (coated vs. uncoated; p<0.005). In control tubing systems without stents platelet activation was less pronounced ( p<0.0001). Antibodies to CD41a and CD42b did not show significant changes. In this model platelet activation detected by flow cytometry and time until stent thrombosis were dependent on stent length and coating. In vitro testing could be useful to optimize stent design and material.

Characterization of adhesive and aggregating functions of pig platelets: comparative studies with human platelets and differential effects of calcium chelation

We have characterized functional responses of pig platelets in citrate-anticoagulated blood and compared results with those of human platelets. Platelet aggregation was induced with known activating agents using citrated platelet-rich plasma. Adhesive and cohesive functions of platelets were evaluated using a perfusion flow system with whole anticoagulated blood (10 min, 800/s). To test the differential sensitivity to free calcium levels, two citrate concentrations (19 mM {standard} vs 13 mM) were used as anticoagulants. The ability of platelets to produce thromboxane A2 was also quantified, using radioimmunoassay. In the presence of 19 mM citrate concentration, pig platelets responded poorly to ADP and collagen, and did not respond to arachidonic acid and U46619. Pig platelets adhered well onto subendothelium, but failed to form aggregates on previously spreaded platelets. At 13 mM citrate concentration, the aggregating responses of pig platelets improved, but some of the aggregation patterns were still reversible. The lower citrate concentration had a dramatic impact on perfusion studies, where the formation of platelet aggregates was restored up to levels found in humans. Thromboxane levels in pig serum were only 20 to 30% of that found in humans. So, pig platelets possess functional properties that clearly differ from human platelets: they are more sensitive to the calcium chelating effects of citrate. Arachidonic acid metabolism and thromboxane amplification of platelet responses seem less preponderant in the pig species.

The In Vitro Effect of Ridogrel on Platelet Function in Normocholesterolaemic and Familial Hypercholesterolaemic Type IIa Subjects

Platelets from familial hypercholesterolaemia type IIa patients are hyperreactive and produce increased amounts of thromboxane A 2. These modifications of platelet function may play an important role in the occurrence of premature atherosclerosis. One approach to the prevention of the thromboembolic complications of atherosclerosis is the use of antiplatelet agents which depress platelet function. Ridogrel, a combined thromboxane synthase inhibitor and thromboxane A 2/prostaglandin endoperoxide receptor blocker inhibits platelet aggregation. This study was designed to investigate the in vitro effect of ridogrel on platelet function in normocholesterolaemic and familial hypercholesterolaemia type IIa subjects. In citrated platelet rich plasma ridogrel significantly inhibited platelet aggregation and thromboxane A 2 production in response to collagen, ADP and arachidonic acid stimulation. In washed platelets ridogrel significantly decreased aggregation and serotonin release. Ridogrel significantly increased cAMP levels in response to thrombin stimulation. In conclusion, ridogrel at low concentrations significantly inhibited the in vitro function of platelets in a dose dependant manner in both normocholesterolaemic subjects and familial hypercholesterolaemia IIa subjects.

Surface-Secreted von Willebrand Factor Mediates Aggregation of ADP-Activated Platelets at Moderate Shear Stress: Facilitated by GPIb but Controlled by GPIIb-IIIa

We previously showed that ADP activation of washed human platelets in plasma-free suspensions supports aggregation at moderate shear stress (0.4-1.6 Nm-2) in Poiseuille flow. Although most activated platelets expressed maximal fibrinogen-occupied GPIIb-IIIa receptors, aggregation appeared to be independent of bound fibrinogen, but blocked by the hexapeptide GRGDSP. Here, we tested the hypothesis that von Willebrand factor (vWF) secreted and expressed on activated platelets mediates aggregation at moderate shear rates from 300 to 1000 s-1 corresponding to shear stresses from 0.3 to 1.1 Nm-2. Relatively unactivated platelets (< 15% expressing prebound fibrinogen) were prepared from acidified citrated platelet rich plasma (cPRP) by single centrifugation with 50 nM stable prostacyclin derivative ZK 36374 and resuspended in Tyrodes-albumin at 5 x 10(4) cells microliter-1. Flow cytometric measurements with monoclonal antibody (mAb) 2.2.9 reporting on surface-bound vWF, and with mAb S12 reporting on alpha-granule secreted P-selectin, showed that 65% and 80%, respectively, of all platelets were maximally activated with respect to maximal secretion and surface expression of these proteins. "Resting" washed platelets exhibited both surface-bound vWF and significant P-selectin secretion. We showed that mAbs 6D1 and NMC4, respectively blocking the adhesive domains on the GPIb receptor recognizing vWF, and on the vWF molecule recognizing the GPIb receptor, partially inhibited ADP-induced aggregation under shear in Couette flow, the degree of inhibition increasing with increasing shear stress. In contrast, mAb 10E5, blocking the vWF binding domain on GPIIb-IIIa, essentially blocked all aggregation at the shear rates tested. We conclude that vWF, expressed on ADP-activated platelets, is at least the predominant cross-bridging molecule mediating aggregation at moderate shear stress. There is an absolute requirement for free activated GPIIb-IIIa receptors, postulated to interact with platelet-secreted, surface bound vWF. The GPIb-vWF cross-bridging reaction plays a facilitative role becoming increasingly important with increasing shear stress. Since aurin tricarboxylic acid, which blocks the GPIb binding domain on vWF, was also found to completely block aggregation in Poiseuille flow, we conclude that it too affects the GPIIb-IIIa interaction.

Comparativein VitroAnalysis of Topical Hemostatic Agents

Hemostatic agents are broadly utilized across the surgical specialties. While specific characteristics desirable for individual applications may vary, these agents generally are expected to aid the patient's coagulation system in the rapid development of an occlusive clot. Six commonly utilized hemostatic agents were quantitatively compared in terms of their ability to mediate platelet aggregation, deposition and activation, and initiate gross clot formation in a series ofin vitrotests. Three types of collagen sponges (Actifoam, Helistat, Instat), microfibrillar collagen (Avitene), a gelatin sponge (Gelfoam), and oxidized regenerated cellulose (Surgicel) were studied. Platelet aggregation: Citrated platelet rich plasma (PRP) was contacted with hemostatic agents both with and without thrombin in a stirred cuvette and the platelet count was measured over 5 min. Platelet deposition: To approximate arterial wounds, PRP was perfused in a controlled manner through hemostatic agents, and the effluent platelet count was measured over time. Platelet activation: ATP secretion from PRP contacted with hemostatic agents in a stirred cuvette was measured at 1 and 5 min. Clot formation: The clotting time of nonanticoagulated whole blood contacted with the hemostatic agents was measured. Materials which depleted platelets most rapidly and effectively in the perfusion system (Avitene, Helistat, and Actifoam) were also the most effective inducers of platelet aggregation and secretion. Clot formation (likely to be platelet dependent) was also more rapid in this group. An overall activity ranking combined the relative performance of each hemostatic agent on the various tests: Actifoam ∼ Avitene > Helistat >> Gelfoam > Instat > Surgicel. The activity ranking generally reflects the materials used in these agents (collagen > gelatin > oxidized regenerated cellulose), as well as their processing (chemical crosslinking in collagen sponges may lower activity). Suchin vitroassessment of the relative platelet and coagulation activities of hemostatic agents might serve as a useful screening tool before investigating properties which require more expensive animal or clinical testing.