Autologous Platelet-poor Plasma Research Articles

Mesenchymal Stromal Cells Derived from Canine Adipose Tissue: Evaluation of the Effect of Different Shipping Vehicles Used for Clinical Administration.

Mesenchymal Stromal Cells (MSCs)-based therapies are rapidly gaining interest in veterinary medicine. Cellular therapy represents a new challenge for practitioners and requires precise coordination between the cell processing laboratory and the veterinary clinic. Cryopreservation is the best method to provide fast, in-time, and long-distance delivery of cells for therapeutic applications. However, potentially toxic cryoprotectants and xenobiotic products make the direct administration of cells impracticable for patients. Alternatively, the cells may be resuspended in a ready-to-use vehicle and shipped to the veterinary clinic. In this study, two nutrient-poor vehicles (physiologic saline and ringer lactate solutions) and two nutrient-rich vehicles (the releasate derived from autologous Platelet Poor Plasma and Platelet Rich Plasma) were tested on adipose tissue-derived canine MSCs (AD-MSCs). AD-MSCs stored for 2, 4, or 24 h in the different media were compared regarding mortality, metabolic activity, and replicative capacity. Furthermore, antioxidant activity and the pattern of expression of genes related to AD-MSCs function were performed following 24 h of storage. The results showed that all the different vehicles preserve cell vitality and replication following short-term storage. In long-term storage, the vehicle and cell density affect cell vitality, proliferation, and gene expression (CCL-2, CXCR-4, and TSG-6). Nutrient-rich vehicles seem better suited to preserve cell functionalities in this contest.

Perilesional injections of human platelet lysate versus platelet poor plasma for the treatment of diabetic foot ulcers: A double-blinded prospective clinical trial.

Diabetic foot ulcer (DFU) is a major cause of morbidity, non-traumatic lower limb amputation in diabetic patients and a high-cost burden on the healthcare system. New therapeutic products are increasingly tested. Platelet-rich plasma (PRP) and human platelet lysate (hPL) are reported to be useful. This trial was conducted to test whether the healing effect of hPL in chronic DFU was due to plasma or platelet lysates in a prospective double-blind design. Autologous PRP was obtained from citrated blood, lysed, and used as drug 1 (active product). The platelet-poor plasma (PPP) was used as a drug 2 (placebo). Ten patients were enrolled in arm 1 and 9 in arm 2. The drugs were injected perilesionally every 2 weeks for a total of sixinjections. Adverse events were recorded until Week 14. The DFUs were scored per the Texas and Wegner systems. No patient showed any major adverse events. Some reported local pain post-injection. Wound healing was achieved in the hPL group in 9/10 of patients at a mean of 35.1 days. In the PPP group, no patient had healed by Day 84. The difference was statistically significant at P < 0.00001. We conclude that autologous hPL is safe and highly effective in healing chronic DFU and is superior to autologous PPP.

Erythrocyte Aggregation in Hyperthermic Hypoxic Conditions Significantly Related to Self‐identified Race

Our purpose was to identify the kinetics of erythrocyte (RBC) reversible aggregation (Agg) during stop flow (SF) experiments as a function of temperature and O2 for RBC from healthy subjects self-identified as Black (B, n=8), Hispanic (H, 4) or Caucasian (C, 4) (48+/- 9 y). Whole blood (EDTA, BioIVT, Westbury, NY), vendor verified to be viral free, and used within 1 week of donation, was centrifuged and the platelet poor plasma (PPP) was recovered. All RBCs were resuspended in autologous PPP, and incubated at specified temperatures (37, 41, 45, 49 C) and O2 (0, 5, 10%, saturating). Flow was initiated in a microchannel system at 0.1-0.2 Re, then stopped while recording the entire microchannel width. The greyscale range of the images showed a significant Fahraeus effect with flow (cell free layer at wall). With SF, as Agg proceeded, the aggregates accumulated within the centerline of the microchannel, increasing the variability of the greyscale range from white at the microchannel wall to black in the centerline. The change in variability indicated kinetics of Agg that occurred over 3 min of recording. The primary data of greyscale vs time (custom software) was fit to a sigmoid (dose response) curve, where the fitted EC50 was the T1/2 for Agg and with the fitted slope (p) indicated the kinetics of Agg. At 37 C the T1/2 was 93 s in 0% O2, 75 s with venous (5%) or arterial (10%) and was not different by group. As temperature increased, the T1/2 became significantly faster in blood from B or H (to 65 s), but not C. Correlation between temperature and the fitted T1/2 were significant in B (slope, R2; -2.9, 0.92), H (-2.7, 0.97) and C (-1.1, 0.6), but the F-test on the slope was significant for B (p=0.02) and H (0.04) not C (0.6). This coincided with a significantly steeper p for B and H. At venous, arterial or saturating O2, the effect of increasing temperature to decrease the T1/2 was much less pronounced, and not different between groups. This data shows that in only hypoxic conditions, with temperatures of 41 C (105.8 F) or higher, RBC from self-identified B or H subjects aggregates more readily than C. These results pertain to our focus of microvascular compromise in thermal burn injury progression, but also clinical conditions of localized cautery to control bleeding, high fevers or malignant hyperthermia.

The Effect of Platelet-Rich Plasma on Endothelial Progenitor Cell Functionality.

Platelets mediate circulating endothelial progenitor cell (EPC) recruitment and maturation, participating in vascular repair, however the underlying mechanism(s) remain unclear. We investigated the effect of platelet-rich plasma (PRP) on the functionality of CD34+-derived late-outgrowth endothelial cells (OECs) in culture. Confluent OECs were coincubated with PRP under platelet aggregation (with adenosine diphosphate; ADP) and nonaggregation conditions, in the presence/absence of the reversible P2Y12 platelet receptor antagonist ticagrelor. Outgrowth endothelial cell activation was evaluated by determining prostacyclin (PGI2) and monocyte chemoattractant protein-1 (MCP-1) release and intercellular adhesion molecule-1 (ICAM-1) membrane expression. Similar experiments were performed using human umbilical vein endothelial cells (HUVECs). Platelet-rich plasma increased ICAM-1 expression and PGI2 and MCP-1 secretion compared with autologous platelet-poor plasma, whereas ADP-aggregated platelets in PRP did not exhibit any effect. Platelet-rich plasma pretreated with ticagrelor prior to activation with ADP increased all markers to a similar extent as PRP. Similar results were obtained using HUVECs. In conclusion, PRP induces OEC activation, a phenomenon not observed when platelets are aggregated with ADP. Platelet inhibition with ticagrelor restores the PRP capability to activate OECs. Since EPC activation is important for endothelial regeneration and angiogenesis, we suggest that agents inhibiting platelet aggregation, such as ticagrelor, may promote platelet-EPC interaction and EPC function.

Xenobiotic-Free Medium Guarantees Expansion of Adipose Tissue-Derived Canine Mesenchymal Stem Cells Both in 3D Fibrin-Based Matrices and in 2D Plastic Surface Cultures.

Mesenchymal stem cells (MSCs) have been recently introduced in veterinary medicine as a potential therapeutic tool for several pathologies. The large-scale in vitro expansion needed to ensure the preparation of a suitable number of MSCs for clinical application usually requires the use of xenogeneic supplements like the fetal bovine serum (FBS). The substitution of FBS with species-specific supplements would improve the safety of implanted cells, reducing the risk of undesired immune responses following cell therapy. We have evaluated the effectiveness of canine adipose tissue-derived stromal vascular fraction (SVF) and MSCs (ADMSCs) expansion in the presence of canine blood-derived supplements. Cells were cultured on traditional plastic surface and inside a 3D environment derived from the jellification of different blood-derived products, i.e., platelet-poor plasma (PPP), platelet-rich plasma (PRP), or platelet lysate (PL). PPP, PRP, and PL can contribute to canine ADMSCs in vitro expansion. Both allogeneic and autologous PPP and PL can replace FBS for ADMSCs culture on a plastic surface, exhibiting either a similar (PPP) or a more effective (PL) stimulus to cell replication. Furthermore, the 3D environment based on homospecific blood-derived products polymerization provides a strong stimulus to ADMSCs replication, producing a higher number of cells in comparison to the plastic surface environment. Allogeneic or autologous blood products behave similarly. The work suggests that canine ADMSCs can be expanded in the absence of xenogeneic supplements, thus increasing the safety of cellular preparations. Furthermore, the 3D fibrin-based matrices could represent a simple, readily available environments for effective in vitro expansion of ADMSCs using allogeneic or autologous blood-products.

Fibrinogen and its Soluble Fibrin as Mediators of Erythrocyte Aggregation

Erythrocyte (RBC) aggregation in blood or in plasma is a reversible phenomenon with an unknown mechanism. However there is evidence that it is induced, at least in part, by fibrinogen. RBC aggregation leads to vascular occlusion or flow diversion and shunting of oxygenated blood. If fibrinogen/fibrin ultimately becomes the major occlusive moiety, as one of us has observed in peri‐burn tissue at 48h post‐burn, the occlusion is likely to be irreversible. Previous studies exploring fibrinogen induced aggregation used Millipore fibrinogen. However, based on our preliminary observation of fibrinogen‐induced aggregation, we propose that the same concentration of fibrinogen across different company brands will induce aggregation of variable extent. Millipore fibrinogen was compared with isolated soluble fibrin‐depleted fibrinogen (FgD) and with soluble fibrin‐rich fibrinogen (FgR) (human). Using a spectrophotometer (Bio‐Rad SmartSpec 3000), focused on wavelengths of 540, 560, and 580nm, corresponding to a peak in oxy, deoxy, and oxygenated hemoglobin state in the RBCs, each fibrinogen isolated was tested at varying concentrations (1, 2, and 4mg/mL) and temperatures (32, 37, and 45°C) with washed human RBC. Across all temperatures and wavelengths, FgR fibrinogen dissolved in phosphate buffered saline (PBS) had a higher percent transmission, which correlates to an increased RBC aggregation, than both Millipore fibrinogen and FgD fibrinogen. Additionally, FgR induced the most aggregation at the highest tested concentration, 4mg/mL. Using this increased aggregation at 4mg/mL of FgR, we then compared to PBS and autologous platelet poor plasma. Across all temperatures, the magnitude of RBC aggregation is higher in plasma than FgR, which induces higher aggregation than PBS alone. The results demonstrated that fibrinogen‐induced RBC aggregation varies with the levels of its soluble fibrin/fibrinogen complex. By extension, these complexes clearly enhance RBC aggregation, intimating an in vivo role particularly in pathologic conditions that increase their levels.

Autologous platelet-poor plasma decreases the bronchial stump necrosis in rat

BackgroundNecrosis of the bronchial stump is a very important trigger for bronchopleural fistula. The administration of local autologous platelet-poor plasma (PPP) could protect the bronchial stump. Materials and methodsLeft pneumonectomy was performed in 25 Sprague-Dawley rats. Animals were randomly assigned to a control group (n=13) and PPP group (n=12). PPP was locally administered on the bronchial stump after pneumonectomy. We analyzed histologic changes in the bronchial stump and messenger RNA expression changes of genes involved in wound repair at 10 and 20 d. ResultsLocal PPP treatment produced a mass of fibrous tissue surrounding the bronchial stump and significantly decreased the presence of necrosis at 20 d. PPP increased the expression of insulin like growth factor 1 at 10 d although it did not reach statistical significance. ConclusionsOur findings indicate that local PPP treatment of the bronchial stump after pneumonectomy decreased necrosis and could have a protective effect on the bronchial stump.

Use of Autologous Fibrin Glue (Platelet-Poor Plasma) in Abdominal Dermolipectomies

Autologous plasma is endowed with properties that speed up healing, hemostasis, and adhesiveness, in addition to growth factors. Through an established protocol, it was possible to isolate thrombin, as well as the platelet-rich plasma (PRP) and platelet-poor plasma (PPP) fractions. The purpose of this study was to analyze autologous use of thrombin and PPP to foster adhesion between an abdominal dermoadipose flap and the aponeurotic surface in abdominal dermolipectomies. The data from 40 patients who underwent abdominal dermolipectomies were analyzed, with 20 patients using thrombin and autologous PPP (Plasma group) and 20 patients with no intervention (Control group). An attempt was made to assess adhesive power by quantifying the serohematic liquid volume gauged during the postoperative days (POD), and also noting the incidence of seroma. Other variables such as age and body mass index (BMI) were also analyzed. The reduction in the aspiration drain debit was statistically relevant only on the first POD in the Plasma group. There was no reduction in the incidence of seroma in these patients. Similarly, age and BMI did not influence these outcomes. The PPP fostered adhesion between the abdominal dermoadipose flap and the aponeurotic surface only on the first POD and had no influence whatsoever on the incidence of seroma. There are few reports on the use of PPP for plastic surgery, particularly the autologous type, opening up possibilities for further research projects to expand its use. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the table of contents or the online instructions to authors www.springer.com/00266.

A predictive biomimetic model of cytokine release induced by TGN1412 and other therapeutic monoclonal antibodies

Human peripheral blood mononuclear cells (PBMC) are routinely used in vitro to detect cytokine secretion as part of preclinical screens to delineate agonistic and antagonistic action of therapeutic monoclonal antibodies (mAbs). Preclinical value of standard human PBMC assays to detect cytokine release syndrome (CRS) has been questioned, as they did not predict the “cytokine storm” that occurred when healthy human volunteers were given a CD28-specific super-agonist mAb, TGN1412. In this article, we describe a three-dimensional biomimetic vascular test-bed that can be used as a more physiologically relevant assay for testing therapeutic Abs. For developing such a system, we used TGN1412 as a model mAb. We tested soluble TGN1412 on various combinations of human blood components in a module containing endothelial cells grown on a collagen scaffold and measured cytokine release using multiplex array. Our system, consisting of whole leukocytes, endothelial cells, and 100% autologous platelet-poor plasma (PPP) consistently produced proinflammatory cytokines in response to soluble TGN1412. In addition, other mAb therapeutics known to induce CRS or first infusion reactions, such as OKT3, Campath-1H, or Herceptin, generated cytokine profiles in our model system consistent with their in vivo responses. As a negative control we tested the non-CRS mAbs Avastin and Remicade and found little difference between these mAbs and the placebo control. Our data indicate that this novel assay may have preclinical value for predicting the potential of CRS for mAb therapeutics.

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

Platelet Function Testing: Auditing Local Practice and Broader Implications

Platelet function testing is a common test procedure used for assessing patients with mucocutaneous bruising and/or bleeding and for monitoring anti-platelet therapy. However, standardization of practice is poorly applied, and experts differ on several aspects of its application. This study reports on a local audit of current practice in consideration of recent reports, expert opinion and current CLSI guidelines. We undertook an assessment of our laboratory test practice for light transmission aggregometry (LTA) as a diagnostic screening process for platelet function, as well as performance of PFA-100 closure times used for screening primary haemostasis. For LTA testing, we wished to assess the validity or otherwise of platelet count adjustments using autologous platelet poor plasma (PPP), as used by some experts and as also recommended by the current CLSI guidelines for performance of platelet aggregation. For PFA-100 testing, we assessed the effect of different blood collection tubes. For most test cases undergoing LTA, platelet count adjustment using autologous PPP resulted in considerable diminution of detectable platelet function using several agonists, and in particular collagen, ADP and epinephrine. These effects could result in differing conclusions regarding the likelihood or severity of a platelet function disorder. For the PFA-100, different blood collection tubes resulted in slightly different closure times that could also potentially influence the conclusion of 'normality' or otherwise for investigated patients. This audit of local practice indicates that the process of platelet count adjustment using autologous PPP provides adverse outcomes related to identification of platelet dysfunction. Accordingly, we recommend that all laboratories validate this practice if used at their facility. For PFA-100 testing, local validation of the normal reference range is required according to local conditions and collection practice. Otherwise, laboratories may inappropriately identify platelet function disorders when these may not exist.

Diagnostic Utility of Platelet Secretion Testing by ATP Release Assays: Results From a Prospective Study of Individuals Referred for Bleeding Disorder Assessments.

Abstract 1314Poster Board I-338 BackgroundPlatelet secretion defects are a common type of inherited platelet disorder. Experts agree that ATP release assays are often helpful to assess for platelet secretion disorders among individuals with suspected bleeding problems. However, the diagnostic utility of ATP release assays has not been established, and it is unclear if the findings correlate with aggregation results. Our goal was to evaluate the incidence and spectrum of platelet release abnormalities in a prospective cohort of individuals referred for testing as part of a bleeding disorder assessment, and describe the diagnostic utility of platelet ATP release and its relationship to aggregation findings. MethodsData were analyzed for 40 healthy controls and 76 patients from a prospective cohort of individuals referred for bleeding disorder assessments. ATP release and light transmission aggregometry (LTA) were assessed by standardized methods, with validated reference intervals (derived by non-parametric analysis), using platelet-rich plasma adjusted to 250 × 109 platelets L−1 with autologous platelet poor plasma. ATP release was assessed using the Chronolog lumiaggregometer, as recommended, and a modified agonist panel (1.6 mM arachidonic acid, 1 uM thromboxane analogue U46619, 100 uM epinephrine, 6 uM epinephrine, 5 ug·mL−1 collagen, 1.25·g mL−1 collagen, 1 U·mL−1 thrombin, and 5 uM ADP). The test sensitivity and specificity for inherited platelet disorders were estimated and the likelihoods of detecting an inherited platelet disorder by ATP release (using different agonists or the agonist panel) were expressed as odds ratios (OR) and 95% confidence intervals (95% CI). Correlations between ATP release triggered by different agonists, and between ATP release and maximal aggregation with the same agonist, were expressed as Pearson correlation coefficients (ρ). ResultsReduced ATP release with one or more agonists was more common among the referred individuals that a hematologist categorized as having a bleeding problem (n = 66; 45% abnormal) than among healthy controls (5% false positives, p < 0.0001). The likelihood of an inherited platelet disorder was high (OR 83, 95% CI 16 - 445) when ATP release was abnormal with one or more agonists, even when LTA results were normal (OR 56, 95% CI 10 - 318). Impaired ATP release with some agonists (e.g. 5 uM ADP and 100 uM epinephrine) could not be detected as the reference intervals did not have a measurable lower limit. Among other agonists, 1 U·mL−1 thrombin had the highest likelihood of detecting impaired ATP release due to an inherited platelet disorder (OR 32 compared to 17- 25 for other agonists).Receiver operator curve analyses indicated that an assessment of ATP release with the full panel, or a more limited panel of 4 agonists, had high specificity and moderate sensitivity for detecting inherited defects in platelet function. The predictive power was improved by considering results as continuous rather than categorical (normal/abnormal) variables, however, this required complex mathematical equations. Most abnormalities in ATP release due to common inherited platelet disorders were detected by a limited number of agonists in the panel. The optimal panel, with minimized false positive and false negative rates (5% and 18% respectively), included: 1 uM thromboxane analogue U46619, 6 uM epinephrine, 5.0 ug·mL−1 collagen, and 1 U·mL−1 thrombin.Among patient subjects and healthy controls, there was a significant correlation (ρ = 0.25 – 0.92; p values < 0.02) between the amount of ATP release induced by most agonists. Maximal aggregation and ATP release responses for each agonist did not correlate significantly among healthy controls, and showed only a weak correlation among patient subjects. ConclusionsLike light transmission aggregometry, ATP release assays have important diagnostic utility for detecting impaired platelet function due to common inherited bleeding disorders. The poor correlation between ATP release and maximal platelet aggregation suggests that these tests are predominantly influenced by different parameters of platelet function. Our data on useful agonists for detecting impaired ATP release may prove helpful to laboratories that wish to optimize testing for common platelet disorders. The knowledge that platelet secretion assays are very helpful for bleeding disorder assessments should be translated into clinical practice. DisclosuresNo relevant conflicts of interest to declare.

No influence of the VAMP8 rs1010 single nucleotide polymorphism on platelet functions in vitro

No influence of the VAMP8 rs1010 single nucleotide polymorphism on platelet functions <i>in vitro</i>

Topical application of autologous blood products during surgical closure following a coronary artery bypass graft☆

Surgical site wound complications are associated with increased cost, morbidity, and mortality following cardiothoracic surgery. Recent publications have advocated the application of autologous blood components as an adjunctive tool to surgical closure during various surgical procedures. The current study was intended to assess the safety and efficacy of the application of autologous platelet rich and platelet poor plasma to the sternal closure and saphenous vein harvest site during closure following a coronary artery bypass graft. A retrospective analysis was performed on 1446 consecutive coronary artery bypass grafting procedures from two surgeons. A patient group receiving topical application of platelet rich and platelet poor plasma during closure of their chest and leg surgical incisions was compared to a patient population receiving standard treatment of care. Forty covariates were collected for each patient included in the study. Propensity scoring was used to adjust for baseline imbalance. Asymptotic logistic regression and exact statistical methods were used to determine the effect of the autologous blood application on infection and drainage of the sternal and leg wounds. One thousand, one hundred and twenty-eight patients had sufficient data to be included in the final analysis, with 571 of these patients receiving the treatment compared to 557 control patients. No treatment-related adverse events were noted and the application process did not significantly affect the operative time. This retrospective analysis of a consecutive series of patients receiving a coronary artery bypass grafting procedure concluded that application of platelet rich and platelet poor plasma significantly reduced occurrences of chest wound infection, chest drainage, and leg wound drainage. This novel therapy merits further investigation.

Evaluation of Autologous Platelet-Poor Plasma Gel as a Hemostatic Adjunct After Posterior Iliac Crest Bone Harvest

To evaluate the hemostatic efficacy of autologous platelet-poor plasma (PPP) gel following posterior iliac crest bone graft harvesting for oral and maxillofacial reconstruction. This was a prospective study of 24 consecutive patients involving 26 posterior iliac crest bone harvests that had bone wax and either 1-gram of bovine microfibrillar collagen or 20 mL of autologous PPP, activated as a gel, used as adjunct hemostatic agents. Compression bulb suction drain was placed into the graft site and drain output recorded every 8 hours for 64 hours. Cost analysis was also undertaken between the 2 methods. Statistical significance between means of each group was determined by Student's t test and found significant for P < .05. There were no statistically significant differences in average drain output between the PPP and MFC groups for each 8-hour interval. There was no statically significant difference in average total drain output between the PPP and MFC groups over the entire 64 hour period. Additionally, unlike the addition of MFC, the addition of PPP added no additional costs to the procedure. PPP gel, when compared with bovine microfibrillar collagen, is an effective and inexpensive adjunct in hemostasis following posterior iliac crest bone harvest.

Platelet aggregation studies: autologous platelet-poor plasma inhibits platelet aggregation when added to platelet-rich plasma to normalize platelet count

Adjusting platelet count (PC) in platelet-rich plasma (PRP) using platelet-poor plasma (PPP) is recommended for platelet aggregation (PA) studies, but it could also affect PA independently of the decrease in PC. Analysis of aggregation tracings from healthy controls showed that PC correlated with PA in 47 diluted-PRPs, but not in 104 undiluted-PRPs. Dilution of 9 PRPs with PPP progressively decreased PA, while dilution of washed platelets with buffer hardly affected PA. Apyrase partially prevented the inhibitory effect of PPP. Therefore, the practice of diluting PRP with PPP to adjust platelet count should be avoided because it artefactually inhibits PA.

Whole blood laboratory model of thrombocytopenia for use in evaluation of hemostatic interventions

This study describes a laboratory model of whole blood (WB) thrombocytopenia established with blood from healthy volunteers. We obtained a mean platelet count of 16 x 10(9)/l (95% confidence interval, 10-22) in WB by repeatedly replacing the platelet-rich supernatant with autologous platelet-poor plasma from the same individual. Thrombelastographic profiles of WB clot formation and WB clot stability were performed in parallel with measurements of WB platelet aggregation response. Thrombocytopenia reduced the maximum rate of WB clot formation, while ex vivo addition of platelets reversed the coagulopathy of thrombocytopenia. Control experiments revealed minimal changes in coagulation factors, distribution of bloods cells, and platelet activation capabilities. The WB model appears useful in research, development, and evaluation of the effects of hemostatic interventions in thrombocytopenia.

Anti-Platelet Properties of Salivary Secretions of the Avian Leech.

IntroductionHaematophages, animals evolved to a bloodsucking lifestyle as their exclusive mode of feeding secrete compounds capable of arresting haemostasis in the host. It is clear that exploitation of host haemostasis is an absolute requirement for the survival of these species. Since the discovery and with the subsequent characterisation and engineering of Hirudin a potent thrombin inhibitor from the European medicinal leech Hirudo medicinalis, attention has been focused on the potential anticoagulant and platelet aggregation inhibitors derived from an array of different species of leech from both the Rhynchobdellid and Arhynchobdellid orders. Haematophagous leeches of the genus Theromyzon sp. of the Rhynchobdellid order, also termed duck leeches, feed directly on the nasal passages, trachea and nictating membranes of migratory birds. We present the novel observation of inhibition of aggregation of human platelets by the salivary secretion extracts of the avian leech Theromyzon tessulatum.MethodsTwelve adult leeches of the species T. tessulatum (total weight 2.828g) were anaesthetised with ethanol vapour. The leeches were severed at the anterior end and a homogenate produced containing salivary gland secretions. The posterior two thirds of the leeches were treated identically to serve as control material. Platelet rich plasma (PRP) was prepared from blood from a normal individual (free from known platelet modifying medicines) mixed 9:1 (v:v) with 0.105 M trisodium citrate in siliconised glass vacutainers. Platelet numbers were adjusted with autologous platelet poor plasma (PPP) to obtain concentrations of approximately 300 × 109/L. Leech extracts (anterior or posterior control) were added to PRP at a ratio of 1:4. Aggregation studies were performed using thrombin (10units/ml), collagen 10μg/ml, Ristocetin (1.5mg/ml) and ADP (5μm/ml).ResultsData from this study shows that platelet aggregation was completely inhibited when stimulated by thrombin, collagen and ADP and partially inhibited (40%) on the addition of ristocetin.ConclusionOur observations contradict the belief that the anti-thrombocyte properties of this species of haematophagous leech are restricted to duck thrombocytes. We suggest the presence of one or more inhibitory molecules acting by various mechanisms including inhibition of vWF and platelet integrin mediated collagen interactions, inhibition of ristocetin mediated vWF and platelet GPIb receptor binding and salivary secretion derived apyrase inhibition of arachidonic acid mediated platelet aggregation.These findings provide conclusive evidence that this blood sucking bird leech has the ability to overcome thrombocyte function in higher vertebrates.

Thrombin generation in patients with cirrhosis: The role of platelets

Coagulation factor defects, thrombocytopenia, and thrombocytopathy are associated with cirrhosis. However, bleeding in patients who have cirrhosis does not entirely correlate with abnormal coagulation tests. Recently, it was shown that because of the concomitant abnormalities of the procoagulant and anticoagulant drives, thrombin generation in plasma patients with cirrhosis is normal when assessed with assays that include thrombomodulin (the main protein C activator). However, thrombin is also generated in vivo as a function of platelets, suggesting that thrombocytopenia and thrombocytopathy might affect thrombin generation in patients with cirrhosis. We addressed this issue using an assay that accounts for the contribution of plasma and platelets. The study showed that platelet-rich plasma with platelets adjusted by dilution of autologous platelet-rich into autologous platelet-poor plasma to a standard count (100 x 10(9)/L) generates as much thrombin in patients with cirrhosis as in controls (1,063 nmol/L vs. 1,167 nmol/L; P value not significant). When platelets were adjusted to correspond to whole-blood counts, patients with cirrhosis generated significantly less thrombin than controls (949 nmol/L vs. 1,239 nmol/L; P < .001). Furthermore, thrombin generation correlated with platelet numbers (rho = 0.50; P < .001). In addition, the amount of thrombin generated as a function of the whole-blood patients' platelet counts increased significantly when the numbers were adjusted to 100 x 10(9)/L (953 nmol/L vs.1,063 nmol/L; P < .001). In conclusion, severe thrombocytopenia may limit thrombin generation in patients with cirrhosis. These findings might justify platelet transfusion in patients with low platelet counts when they bleed spontaneously or before undergoing surgery or liver biopsy. Controlled clinical trials supporting this indication are warranted.

3. Internationales Symposium: „Autologe Transfusion - Von der Euphorie zur Ratio: Praktisches Handeln aus wissenschaftlicher Sicht” (Teil III)Does Hemodilution Enhance Coagulability?

Recent publications reported enhanced coagulability in hemodilution determined by TEG. In contrast, earlier reports have shown prolongation of in-vivo bleeding time in anemia. In order to take a closer look at this discrepancy undiluted and diluted anticoagulated blood samples (20 % with saline solution, hydroxyl-ethyl starch 6 % (HES), autologous platelet poor plasma (PPP)) were investigated by TEG (n = 10), ball (n = 10), and hook coagulometer (n = 15) as well as tests simulating primary hemostasis ex vivo (Platelet Function Analyzer PFA-100, n = 10). Dilution with plasma changed TEG parameters in a way, when started by recalcification of the blood sample, which is characteristic of enhanced coagulability (r decreased in all and k in 8 of 10 samples, maximal amplitude increased in 9 out of 10). With HES, changes in TEG parameters mainly indicated reduced coagulability (k increased in 7 out of 10, MA decreased in 10 out of 10). When the coagulation was additionally activated by PTT reagent (InTEG) the TEG parameters also mainly showed hypocoagulation with the three dilution solutions. Coagulation times with ball and hook coagulometers were significantly prolonged by dilution especially with saline (+ 25 % and + 17 %, p < 0.001). Dilution always significantly (often abnormally) prolonged closure time in PFA-100 (saline + 41 +/- 18 %, PPP + 37 +/- 20 %, HES + 69 +/- 24 %) demonstrating disturbance of primary hemostasis, particularly with HES. From the results obtained it can be concluded that the changes in the classical TEG (without addition of PTT-reagent), suggesting an enhanced coagulability, may be caused methodically as they are also found with autologous PPP. On the other hand, a disturbance of the primary hemostasis in hemodilution has to be taken into account from the results seen with the PFA-100 and a number of published data.