Induced Platelet Agglutination Research Articles

Reversible Thrombocytopenia of Functional Platelets after Nose-Horned Viper Envenomation Is Induced by a Snaclec.

Profound and transient thrombocytopenia of functional platelets without bleeding was observed in patients envenomed by Vipera a. ammodytes (Vaa). This condition was rapidly reversed by administration of F(ab)2 fragments of immunoglobulin G targeting the whole venom, leaving platelets fully functional. To investigate the potential role of snake venom C-type lectin-like proteins (snaclecs) in this process, Vaa-snaclecs were isolated from the crude venom using different liquid chromatographies. The purity of the isolated proteins was confirmed by Edman sequencing and mass spectrometry. The antithrombotic effect was investigated by platelet agglutination and aggregation assays and blood coagulation tests. Using flow cytometry, the platelet activation and binding of Vaa-snaclecs to various platelet receptors was analyzed. Antithrombotic efficacy was tested in vivo using a mouse model of vascular injury. Two Vaa-snaclecs were purified from the venom. One of them, Vaa-snaclec-3/2, inhibited ristocetin-induced platelet agglutination. It is a covalent heterodimer of Vaa-snaclec-3 (α-subunit) and Vaa-snaclec-2 (β-subunit). Our results suggest that Vaa-snaclec-3/2 induces platelet agglutination and consequently thrombocytopenia by binding to the platelet receptor glycoprotein Ib. Essentially, no platelet activation was observed in this process. In vivo, Vaa-snaclec-3/2 was able to protect the mouse from ferric chloride-induced carotid artery thrombosis, revealing its applicative potential in interventional angiology and cardiology.

Platelet depletion enhances lethal, hemorrhagic and myotoxic activities of Bothrops asper snake venom in a murine model

Platelets play key roles in hemostasis, inflammation, immune response, and tissue repair. Although it is known that viperid snake venoms induce thrombocytopenia and platelet hypoaggregation, the roles of these effects in the overall outcome of envenoming are poorly known. This study aimed to assess the effect of platelet depletion on several toxic activities induced by the venom of the Central American viperid snake Bothrops asper in a mouse model. A profound thrombocytopenia was induced in mice by the administration of aspercetin, a C-type lectin-like protein that induces platelet agglutination and drop in platelet counts, while a control group was treated with saline solution instead. Upon envenoming, animals rendered thrombocytopenic developed a higher extent of local and systemic hemorrhage and local myonecrosis, as compared to control envenomed mice. In addition, the median lethal dose (LD50), determined by the intraperitoneal route, was significantly lower in thrombocytopenic mice, underscoring a higher toxicity of venom in these conditions. No difference in the value of LD50 between the two groups was observed when using the intravenous route of injection, and no difference was observed in the magnitude and time-course of footpad edema. Skeletal muscle regeneration was assessed 14 days after venom injection in muscle. Both experimental groups showed a similarly poor regeneration, suggesting that platelets do not play a key role in the regenerative process in these experimental conditions. Results indicate that depletion of platelets increases hemorrhagic and myotoxic effects, as well as overall toxicity, of B. asper venom, implying that platelets play a protective hemostatic role in this model of envenoming.

Bitiscetin-3, a Novel C-Type Lectin-like Protein Cloned from the Venom Gland of the Viper Bitis arietans, Induces Platelet Agglutination and Inhibits Binding of Von Willebrand Factor to Collagen.

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and—at least bitiscetin-1—induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein—bitiscetin-3—were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and β share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3β were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit—which differs from that of both bitiscetin-3 subunits—the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and β-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.

Dubin Johnson Syndrome associated with defective ristocetin – induced platelet agglutination

Background: This is the first reported case of platelet aggregation defect in association with Dubin-Johnson Syndrome. Case presentation: We reported a 14 months old female infant with recurrent attacks of mild to moderate ear and nose bleeding and moderate amount of subarachnoid hemorrhage with ristocetin induced platelet agglutination other than Von Willebrand Factor disease and Bernard–Soulier syndrome. The patient also had fluctuating bilirubin level (Maximum of 15 mg/dl) and remaining liver function tests were normal; the patient diagnosed as Dubin Johnson Syndrome by liver biopsy. Conclusions: Dubin-Johnson Syndrome may be associated with platelet aggregation defect.

COVID-19-associated pseudothrombocytopenia.

COVID-19-associated pseudothrombocytopenia.

Targeting shear gradient activated von Willebrand factor by the novel single-chain antibody A1 reduces occlusive thrombus formation in vitro.

Intraluminal thrombus formation precipitates conditions such as acute myocardial infarction and disturbs local blood flow resulting in areas of rapidly changing blood flow velocities and steep gradients of blood shear rate. Shear rate gradients are known to be pro-thrombotic with an important role for the shear-sensitive plasma protein von Willebrand factor (VWF). Here, we developed a single-chain antibody (scFv) that targets a shear gradient specific conformation of VWF to specifically inhibit platelet adhesion at sites of shear rate gradients (SRG) but not in areas of constant shear. Microfluidic flow channels with stenotic segments were used to create SRG during blood perfusion. VWF-GPIbα interactions were increased at sites of SRG compared to constant shear rate of matched magnitude. The scFv-A1 specifically reduced VWF-GPIbα binding and thrombus formation at sites of SRG but did not block platelet deposition and aggregation under constant shear rate in upstream sections of the channels. Significantly, the scFv A1 attenuated platelet aggregation only in the later stages of thrombus formation. In the absence of shear, direct binding of scFv-A1 to VWF could not be detected and scFVA1 did not inhibit ristocetin induced platelet agglutination. We have exploited the pro-aggregatory effects of SRG on VWF dependent platelet aggregation and developed the shear gradient-sensitive scFv-A1 antibody that inhibits platelet aggregation exclusively at sites of SRG. The lack of VWF inhibition in non-stenosed vessel segments places scFV-A1 in an entirely new class of anti-platelet therapy for selective blockade of pathological thrombus formation while maintaining normal hemostasis.

Superiority of the Rapid Von Willebrand Factor (VWF) VWF:GPIbR and VWF:GPIbM Assays in Type 2A, 2B and 2M Von Willebrand Disease

A complete set of rapid activity and classical von Willebrand factor (VWF) assays for Willebrand disease (VWD) diagnosis was used in the present study to characterize VWD type 1, 2A, 2B and 2M patients due to mutations in the A1, A2 and A3 domains. The VWF:RCo/VWF:Ag, VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF:Ag ratios at cuttt off value of 0.7 separated VWD type 1 and LowVWF from VWD type 2. The results from the Brno cohort of VWD 2A patients with the G1579R mutation in the A2 domain in sixteen affected member from five families and in one case with the G1609R in the A2 domain revealed that the VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF: Ag ratios are marked decreased (range 0.03-0.27) to a similar degree as compared to VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios (range 0.03-0.27) due to the proteolytic loss of large and intermediate VWF multimers. The results in VWD 2B patients due to gain of ristocetin induced platelet agglutination (RIPA) function mutations R1306W, R1308C and R1341 in the A1 domain demonstrated that the ratios for VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF:Ag as compared to VWF:RCo/VWF:Ag ratio were markedly decreased in VWD 2B, whereas the VWF:GPIbM/VWF:Ag ratio was somewhat higher (range 0.32 to 0.36) in VWD 2M. VWD 2M patients due to loss of RIPA function mutation R1359K in the A1 domain are featured by decreased VWF ratios for WVF:RCo/Ag and VWF:GPIbR/Ag, but less decreased for the VWF:GPIbM/Ag ratio and normal VWF ratio for VWF:CB/Ag ratio the need to retain the VWF:CB assay to make a correct diagnosis of VWD 2M for its differentiation from VWD type 1. The G1415D mutation in the A1 domain is featured by decreased RIPA and decreased VWF:RCo/VWF:Ag ratio (VWD 2M) but normal values for VWF:CB/VWF:Ag, VWF:GPIbM/VWF:Ag and VWFGPIbR/VWF:Ag ratios consistent with VWD 2M. Double heterozygous P1266L/V1278I mutation in two patients and heterozygous E1292D/WT mutation in three patients in the A1 domain were diagnosed as VWD 2M or 1M associated with a secretion defect (SD). The Platelet Function Analyzer Closure Times (PFA-CT) are strongly prolonged in VWD 2A, 2B and 2M. and moderately prolonged between the upper limit of normal to 300 seconds in heterozygous mutated VWD type 1 patients.

Diagnosis and management of von willebrand disease in Spain

The correct diagnosis and classification of von Willebrand disease (VWD) is difficult because of the variability of its clinical expression and limitations of laboratory methods. However, correctly diagnosing VWD is important for therapy and genetic counselling. A survey related to the referred VWD patients in Spain revealed local diagnostic problems in at least one third of the cases of VWD. Consequently, a Spanish multicenter study was carried out in which a cohort of 556 patients from 330 families was analyzed centrally. VWD was confirmed in 480 patients. Next generation sequencing (NGS) of the whole coding von Willebrand factor gene ( VWF ) was carried out in all recruited patients, compared with the phenotype, and a final diagnosis was established. A total of 238 different VWF mutations were found: 154 were not included in the Leiden Open Variation Database (LOVD). Of the patients, 467 were found to have a VWF mutation/s. A good phenotype/genotype association was estimated in 94.6% of the cases. One hundred and eighty patients had two or more mutations. Occasionally a predominant phenotype masked the presence of a second abnormality. One hundred and sixteen patients presented with mutations that had previously been associated with an increased VWF clearance. Ristocetin induced platelet agglutination (RIPA) unavailability, central phenotypic results disagreement and difficult distinction between severe type 1 and type 3 VWD prevented a clear diagnosis in 74 patients. A local/central disagreement over diagnosis occurred in 42.3% of the whole cohort. The NGS study facilitated an appropriate classification in 60 of them. Based on these results, a proposal for a change in the VWD diagnostic paradigm, suggesting the inclusion of the analysis of VWF at the initial diagnostic process, has been made. An extension of this project to include 400 new VWD patients has begun. A diagnostic strategy has been developed, and a Spanish consensus guideline for optimal treatment of VWD was developed in Spain. Desmopressin (DDAVP) is the treatment of choice in responsive VWD patients. VWF concentrates (VWF/FVIII) are used in individuals unresponsive to DDAVP, when DDAVP is contraindicated, or in VWD types 2B and 3, and for long term management needs (e.g., post major surgery).

Evaluation of an heterogeneous group of patients with von Willebrand disease using an assay alternative to ristocetin induced platelet agglutination

Diagnosis of von Willebrand disease (VWD) type 2 usually relies on the discrepancy between the von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) and VWF antigen (VWF:Ag). Type 2B patients can be discriminated from other qualitative VWD variants by using ristocetin-induced platelet agglutination (RIPA) test. The major limitation of RIPA isthe requirement of fresh blood sample. In this study, we evaluated the VWF gain-of-function mutant GPIb binding (VWF:GPIbM) and VWF:RCo assays to investigate whether the VWF:GPIbM/VWF:RCo ratio was able to identify the type 2B variant among an heterogeneous VWD population, previously characterized following the ISTH-SSC guidelines. Seventy-six VWD patients and 31 healthy subjects were evaluated by using VWF:Ag, VWF:RCo, and VWF:GPIbM assays. The mean (minimum-maximum values) VWF:GPIbM/VWF:RCo ratio was higher in type 2B patients (2.53, 0.84-6.11) than in healthy controls (1.05, 0.87-1.34), type 1 (0.85, 0.51-1.15), 2A (1.20, 0.36-2.82), and 2M (1.07, 0.91-1.38) (P<0.0001). Type 2B variants were divided into four groups (A, B, C, and D) according to their different multimeric patterns. The mean value of the VWF:GPIbM/VWF:RCo ratio in the four groups showed an increasing trend from group A (1.08) to D (3.69), proportional to the loss of high molecular weight multimers. Among 32 type 2B patients, previously diagnosed with RIPA, 8 (mainly with a type I New York/Malmö phenotype) were not confirmed using the VWF:GPIbM/VWF:RCo ratio. Whenever the RIPA test is not feasible, the VWF:GPIbM/VWF:RCo ratio might help to identify severe type 2B VWD patients.

Laboratory Assessment of Anti-Coagulant Properties of a Von Willebrand Factor Targeted Aptamer

Introduction: Many cardiovascular procedures including percutaneous coronary intervention, coronary bypass surgery, and peripheral vascular surgery require antithrombotic therapy during the procedure. Heparin is frequently used, due to low cost, ease of monitoring and reversibility with protamine. Antiplatelet agents such as aspirin and clopidogrel are also frequently used but these agents are not reversible. Alternative reversible antithrombotic strategies that interfere with platelet-mediated thrombosis are needed. We are investigating the use of a reversible agent that is specific for von Willebrand factor (VWF) for use during cardiovascular procedures.We previously developed an antidote-controlled 80 oligonucleotide RNA aptamer Ch-9.14-T10 (T10). (Nimjee, S.M., Lohrmann, Wang, H, et al. Molecular Therapy 2012; 2:391-397). T10 bound to VWF with high affinity and inhibited thrombosis in a murine carotid artery damage model. The aptamer also greatly increased bleeding in mice that were surgically challenged by tail transection and blood loss decreased when complementary antidote oligonucleotides were administered. In addition, T10 inhibited primary hemostasis in the PFA-100 assay and this inhibition was completely reversed by the antidote. This study further characterizes the T10 aptamer, as well as a truncated version of the T10 aptamer, using clinical laboratory assays that would be used to monitor antithrombotic therapies during cardiovascular procedures.Methods: We initiated our study with T10 and then developed a truncated version, T25, a 60 oligonucleotide with which we performed additional investigations. We recruited normal donors for analysis of aptamer and antidotes. All participants had normal platelet counts, VWF activity and antigen levels, and were not taking antiplatelet agents. Laboratory tests included: ristocetin induced platelet agglutination (RIPA), VWF activity, ristocetin cofactor activity, cone and plate(let) analyzer, thromboelastography (TEG) and thromboelastometry (ROTEM).Results: We first determined the effects of the VWF aptamers on RIPA, VWF activity, and ristocetin cofactor activity to confirm functional inhibition of VWF. We found that T10 (500-750 nM) and T25 (250-750nM) completely inhibited RIPA depending on the donor. T25 also inhibited VWF activity with a Ki of 160-320 nM and ristocetin cofactor activity with an IC50 of 3000 nM. Employing a cone and plate(let) analyzer we found that T25 (750 nm) reduced platelet adhesion by 33-49% on a polystyrene surface under arterial flow conditions (1800 s-1, for 2 minutes). The aggregate size was also reduced by 30%.As ACT and TEG are frequently used during cardiovascular procedures, we performed several measures with our VWF aptamer. T10 had no effect on the ACT (730-2000nM), TEG (1600 nM), or ROTEM (750 nM). Standard methods were used for the TEG (Kaolin activation followed by CaCl2) and ROTEM (EXTEM assay) Topf et al. reported a modified ROTEM protocol where they found that incubating citrated whole blood with ristocetin decreased the clot strength as measured by the maximum amplitude (Thromb.Haemost 2011; 105:1091). We reasoned that an aptamer that bound to VWF would inhibit the effect of ristocetin on the maximum amplitude. We found that T25 did inhibit the effect of ristocetin and titration studies showed that 200-400 nM T25 was needed to restore the maximum amplitude to at least 80% of its original value. An example of the ROTEM tracings is shown in figure 1.Conclusion: The VWF-specific aptamer T25 can be detected by several clinical assays, including the RIPA, VWF activity assays, and the cone and plate(let) analyzer. It is not detectable by an ACT or standard TEG or ROTEM, however. We are able to detect the aptamer using a modified ROTEM and are further defining the adaptability of this assay to facilitate monitoring during cardiovascular procedures.Citrated whole blood from was incubated with buffer or ristocetin (0.7 mg/ml) or ristocetin and T25 (300 nM) as indicated. Clotting was initiated with EXTEM (tissue factor pathway activation) and CaCl2. [Display omitted] DisclosuresBecker:Regado Biosciences: Membership on an entity’s Board of Directors or advisory committees.

The Collagen-binding Integrin α2β1 Is a Novel Interaction Partner of the Trimeresurus flavoviridis Venom Protein Flavocetin-A

Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.

Successful Outcome of Two Pregnancies in a Woman with Bernard Soulier Syndrome

Abstract 4676 INTRODUCTION:Bernard Soulier Syndrome (BSS) is a rare qualitative and quantitative autosomal recessive platelet disorder molecularly characterized by defective GPIb-IX-V membrane glycoprotein that is related to platelet adhesion and interaction with both Von Willebrand factor and endothelium. It is usually expressed by an enlarged bleeding time and decreased yet giant sized platelets, and can cause severe hemorrhagic complications during pregnancy. CASE REPORT:We present two successful pregnancies and labour management on a 33-year-old woman diagnosed in her childhood with BSS. Ristocetin induced platelet agglutination test and membrane glycoprotein study were performed to get the diagnosis. Furthermore, in the last year we have completed the diagnosis with platelet receptor expression flow citometry analysis, finding out depleted antiGPIbα and antiGPIX antibodies. Changes which prevent GPIbα for correct expression and processing were observed. Also integrin αIIbβ3 levels were risen due to the big sized platelets. A PCR product was obtained from genomic DNA using the GPIbα oligonucleotide pair; sense and antisense, and direct DNA sequencing of the amplification was performed in a model ABIprism 377 DNA sequencer (Perkin-Elmer Cetus). The direct sequencing of the sense strand of the 5x fragment shows in the patient the simultaneous presence of G and C nucleotids in 688 position, and the T and A nucleotids in 715 position. These punctual mutations give Ala200Pro substitutions and Cys209Ser in heterozygosis in mature peptide, respectively, Figure 1. The Ala200Pro mutation has not been reported in others cases of Bernard-Soullier, previously. The Cys209Ser has been described in other two Spanish patients: one in homozygosis and the other in heterozygosis associated to the mutation.Our patient presented hemorrhagic symptoms from 18 month old with an average of 40×103/μ l platelet count and increased platelet volume who required multiple platelet transfusions during her childhood. In the menarche main hemorrhagic manifestations consisted on methrorragia and epistaxis which were controlled with tranexamic acid. At the age of 26 a spontaneous ovarian cyst rupture required urgent hospital admission and two platelet pool transfusion. On December 2007 she consulted for epistaxis, and on September 2008 cesarean delivery was planned at 38th week of pregnancy; platelet count was 40×103/μ l, antiplatelet antibodies were negative for membrane glycoproteins and antiHLA-1 was positive against 98% donors tested. As a result, an HLA-compatible donor was searched, and two platelet apheresis were transfused one hour before surgery and every 12 hours after the procedure. The next 4 days one apheresis every 48 hours were transfused with no hemorrhagic symptoms. On January 2010, she presented with her second pregnancy; the patient had severe anemia, with high transfusion requirements; neither hemorrhage or hemolysis was detected, and even the possibility of a Munchausen syndrome was questioned. Unplanned caesarean was performed at 32th week because of the clinical instability of the patient, and this time we could not find on time an HLA-compatible donor, so two random platelet pools were transfused one hour before surgery and one platelet apheresis every day during 4 days after caesarean deliver, without any hemorrhagic complication. The newborn was asymptomatic with normal platelet count. The patientxs haemoglobin level returned to normal after five days of delivery. CONCLUSIONS:We present the successful labour management of two consecutive pregnancies on a young woman diagnosed with BSS. The low prevalence of this syndrome (less than one case per million population) explains the absence of well-established protocols for the management of pregnancy in this disease. Up to date there are only twelve reported cases of pregnancy and delivery in BSS patients, most of them presenting with severe hemorrhagic complications. Correct platelet transfusion is the mainstay of treatment, and screening for anti-platelet antibodies study is mandatory due to its high prevalence in these patients. Disclosures:No relevant conflicts of interest to declare. [Display omitted]

Low VWF:RCo In Subjects with VWF Polymorphisms D1472H and P1467S Due to Decreased Binding of Ristocetin to the VWF A1 Domain

AbstractAbstract 2208Diagnosis of von Willebrand disease (VWD) currently relies on two assays of von Willebrand factor (VWF), the VWF antigen ELISA (VWF:Ag) and the VWF ristocetin cofactor activity assay (VWF:RCo). The latter exploits the capacity of ristocetin to induce VWF – platelet interactions as a measure of VWF function. Ristocetin, however, is a non-physiologic agonist as shear stress is the physiologic stimulus inducing VWF to bind platelets in vivo. Recently we have reported that the VWF:RCo/VWF:Ag ratio is decreased in individuals with an A1 domain polymorphism, D1472H. The lack of bleeding in subjects with this polymorphism suggests D1472H does not create a physiologic defect in VWF – platelet interactions. D1472H is directly adjacent to a known ristocetin-binding area in the VWF A1 region (Leu 1457 – Pro 1471), supporting the hypothesis that D1472H affects the ability of ristocetin to bind VWF. Similarly, a heterozygous sequence change leading to P1467S (located in the same ristocetin binding domain) resulted in an undetectable VWF:RCo but no bleeding symptoms were noted in affected subjects. To further investigate the cause of this observation, we developed a method to study the binding of ristocetin to VWF directly. Maleic anhydride microtiter plates were used to capture ristocetin via its amine groups. A VWF source, either plasma or recombinant VWF (rVWF), was then added, wells washed, and VWF binding detected using anti-VWF antibodies. Both plasma and rVWF bound to the captured ristocetin similarly with ristocetin plating concentrations ranging from 0.01 to 1 mg/ml. Specificity of ristocetin dependent VWF binding was confirmed, as preincubation of ristocetin with rVWF decreased binding of rVWF to immobilized solid-phase ristocetin. No detectable binding was present for a full length rVWF construct with the entire A1 loop deleted (del 1242–1478) or a construct missing part of the A1 loop (del 1392–1402). VWF binding to ristocetin was inhibited by both monoclonal and polyclonal antibodies directed against the VWF A1 loop. VWF A1 loop constructs with the A1 domain polymorphisms D1472H and P1467S showed decreased binding to ristocetin when compared to a wild-type A1 loop construct. Wild-type A1 loop binding to ristocetin in our assay was 0.98 while 1472H A1 loop binding was reduced at 0.77 (p<0.001 compared to wild-type) and 1467S A1 loop binding was 0.45 (p<0.001 compared to wild-type). No difference was seen when full length VWF constructs with these polymorphisms were studied, perhaps due to other ristocetin recognition sequences present in VWF as reported by Scott and colleagues (JBC 1991). Binding of VWF to vancomycin, a glycopeptide antibiotic similar to ristocetin that does not induce platelet agglutination, was normal. The ristocetin-specific interaction with VWF may require a specific structural conformation of the A1 loop. These results suggest that VWF A1 domain polymorphisms, or the disruption of the entire A1 loop, have the ability to interfere with VWF – ristocetin interactions independent from VWF – platelet interactions. Clinical assays of VWF function that depend on ristocetin may therefore provide misleading results for subjects with VWF polymorphisms affecting ristocetin binding. Disclosures:Montgomery: GTI Diagnostics, Inc: Consultancy.

Formation of methionine sulfoxide by peroxynitrite at position 1606 of von Willebrand factor inhibits its cleavage by ADAMTS-13: A new prothrombotic mechanism in diseases associated with oxidative stress

An enhanced formation of reactive oxygen species and peroxynitrite occurs in several clinical settings including diabetes, coronary artery disease, stroke, sepsis, and chronic inflammatory diseases. Peroxynitrite oxidizes methionine and tyrosine residues to methionine sulfoxide (MetSO) and 3-nitrotyrosine (NT), respectively. Notably, ADAMTS-13 cleaves von Willebrand factor (VWF) exclusively at the Tyr1605–Met1606 peptide bond in the A2 domain. We hypothesized that peroxynitrite could oxidize either or both of these amino acid residues, thus potentially affecting ADAMTS-13-mediated cleavage. We tested our hypothesis using synthetic peptide substrates based on: (1) VWF Asp1596–Ala1669 sequence (VWF74) and (2) VWF Asp1596–Ala1669 sequence containing nitrotyrosine (VWF74-NT) or methionine sulfoxide (VWF74-MetSO) at position 1605 or 1606, respectively. The peptides were treated with recombinant ADAMTS-13 and the cleavage products analyzed by RP-HPLC. VWF74 oxidized by peroxynitrite underwent a severe impairment of its hydrolysis. Likewise, VWF74-MetSO was minimally hydrolyzed, whereas VWF74-NT was hydrolyzed slightly more efficiently than VWF74. Oxidation by peroxynitrite of purified VWF multimers inhibited ADAMTS-13 hydrolysis, but did not alter their electrophoretic pattern nor their ability to induce platelet agglutination by ristocetin. Moreover, VWF purified from type 2 diabetic patients showed oxidative damage, as revealed by enhanced carbonyl, NT, and MetSO content and was partially resistant to ADAMTS-13 hydrolysis. In conclusion, peroxynitrite may contribute to prothrombotic effects, hindering the proteolytic processing by ADAMTS-13 of high-molecular-weight VWF multimers, which have the highest ability to bind and activate platelets in the microcirculation.

Coagulation-Related Effect of Bortezomib Treatment in Patients with Relapsed or Refractory Multiple Myeloma.

Proteasomes inhibitors have been recently identified as potential agents, which can influence processes leading to thrombosis. We have previously reported a reduced incidence of deep vein thrombosis (DVT) in 69 patients enrolled in Total Therapy III and treated with (B), dexamethasone, thalidomide, cisplatin and doxorubicin with prophylactic anticoagulation. Patients with relapse MM, not previously exposed to B with normal baseline coagulation parameters (PT, PTT fibrinogen and platelet count) were eligible for the study. Except for the two PCR-based genotyping assays Factor V Leiden and Prothrombin Gene, which were assessed only at enrollment, in table one are listed coagulation factors serially assessed at baseline and within 1 hour after the first dose of B on day 1 and 4 of the first treatment cycle. Platelet Aggregation, with epinephrine, collagen, arachidonic acid, ADP and P- selectin by flow cytometry, were obtained at same time points.Results: Median values of coagulation factors pre and post B infusion are shown in table 1. Platelet count on platelet rich plasma (PRP) decreased after each B treatment (204.1±69.2 × 103 platelets/μL versus 160.8±50.3 × 103 platelets/μL). Platelet aggregation in PRP induced by 10 μM ADP was reduced by 20% and 29% in post-B on day one and four with p-values of 0.033 and 0.009 rispectively. Ristocetin induced platelet agglutination and epinephrine-induced aggregation were both negatively affected by B administration, p=0.0077 and p=0.034. Platelet aggregations elicited by collagen and arachidonic were not affected by B P-selectin expression on platelet surface was overall decrease with each agonist after B administration however no statistical differences were observed. In conclusion this pilot study has shown that even a short in vivo B exposure can significantly impaired platelet number and function and could explain clinical observations.Table 1TestDay 1 Pre-dose MedianDay 1 Post-dose MedianDay 4 Pre-dose MedianDay 4 Post-dose MedianPT (sec)13.413.8513.5514.4PTT (sec)17.3526.226.9525.5Fibrinogen (mg/dL)385360386341.5Protein C (70%–160%)114102120115Protein S (65–140%)89859292.5Antithrombin II (190–127%)9897.5100.5102APC Ration2.3452.332.32.22T.T. (<20s)18.217.917.719.45D-Dimer (ug/mL)0.560.50.50.59Homocysteine (umol/L)8.778.998.698.13

Studies with the Nanobody That Detects the Gain-of-Function of von Willebrand Factor in a Cohort of Patients with Type 2B von Willebrand Disease: Correlation with Platelet Count, VWF Multimers and Molecular Defects.

Background: Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder caused by abnormal von Willebrand factor (VWF) that displays increased affinity to the platelet glicoprotein 1b alpha (GpIba) and is due to a group of mutations clustered within VWF A1 domain. Such an enhanced 2BVWF-GpIba binding usually result in loss of large VWF multimers and moderate-mild thrombocytopenia. A llama-derived antibody fragment (AuVWFa11) recognizing the GpIba-binding conformation has been recently developed (Blood 2005; 106:3035).Aims and design of the study: to further explore the usefulness of AuVWFa11 in type 2B diagnosis, we have prospectively tested AuVWFa11 in our cohort of 16 patients previously characterized by platelet count, VWF multimers and mutations.Methods: Data of platelet count with mean platelet volume (MPV) and morphologic evaluation of the blood smear to search for giant platelets or aggregates were associated with the history of physiologic or pathologic stress conditions such as pregnancy, infections, surgery or use of DDAVP. All patients were diagnosed by ristocetin induced platelet agglutination (RIPA) in the Platelet Rich Plasma (PRP), ristocetin cofactor activity (VWF:RCo) with VWF antigen (VWF:Ag), multimeric structure of VWF. Mutations within VWF A1 domain were searched for and confirmed by sequencing exon 28. AuVWFa11 was tested in 40 normal individuals (expressed as % of active VWF in normal pool plasma =0.70±0.13) and in type 2B.Results: Data (mean ± SD) of the AuVWFa11 tested in the 16 patients with type 2B VWD are correlated with the main phenotypic data and genotype (Table1). Platelet count < 140,000 was found at baseline in only 3/16 (%), but was observed after stress conditions in 12/16 cases (%); no reduced platelet counts was found in 4/16 patients (%) from two different families (R1308L, R1341Q). An increased MPV was found in 12 cases but giant platelet and aggregates in only 1 case. Activated VWF as tested by AuVWFa11 was positive in all but 3 (R1308L) cases, with values ranging from 2 to 6 times higher than normal controls: values > 3 correlate with loss of large VWF multimers and mild-moderate thrombocytopenia.Conclusions: The AVWF11a can show activated VWF in most type 2B VWD patients, especially when 2B VWF mutants induce significant loss of large multimers and thrombocytopenia. Therefore AuVWF11a can be a useful additional tool in the diagnosis of type 2B VWD.Table 1Mutation (n)RIPA (mg/ml)VWF.Ag (U/dL)Plat Count (×10^9/L)MPV (micron^3)Loss of HMW MultAuVWFa11 (ratioNPP)R1306W (5)0.6540±9165±3910.3±2.3YES3.7±1.5R1308C (3)0.7253±16163±6111.5±1.9YES3.3±2.3R1308L (3)0.5048±13341±1048.1±3.1NO0.5±0.2I1309V (1)0.4011522211.8PARTIAL2.1V1316M (2)0.5032±7119±309.2±2.4YES4.4±0.1P1337L (1)0.50482229.5PARTIAL1.3R1341Q (1)0.67434229.9PARTIAL2.9

An Association of Candidate Gene Haplotypes and Bleeding Severity in von Willebrand Disease (VWD) Type 2 Pedigrees.

We analyzed the association of bleeding severity with eight candidate gene haplotypes within pedigrees of twelve index cases of VWD type 2 (six type 2M, three type 2A and three type 2B), using a covariance components model for multivariate traits (Mendel 5.5: QTL Association). In addition to the 12 index cases, these pedigrees included 58 affected and 55 unaffected relatives, as characterized by plasma Ristocetin cofactor (VWF:RCo), levels, VWF antigen (VWF:Ag) levels, ristocetin induced platelet agglutination (RIPA), VWF multimeric analysis and specific mutations of VWF. A bleeding severity score was derived from a detailed questionnaire. The severity of bleeding episodes was ranked from 0 to 5 in each of eleven bleeding categories: epistaxis, cutaneous symptomatic bleeding, bleeding from minor wounds, oral cavity bleeding, gastrointestinal bleeding, bleeding associated with tooth extraction, surgery, postpartum hematoma, muscle hematoma, hemarthrosis, and menorrhagia. Donors were genotyped using specific fluorescent-labeled oligonucleotides in a melting curve analysis (LightTyper, Roche, Inc.). VWF:RCo levels, followed closely by VWF:Ag levels, had the strongest influence on bleeding severity score. ITGA2 promoter haplotype -52T and ITGA2B haplotype 1 (Ile843) were each associated with increased bleeding severity scores (p=0.039 and p = 0.024, respectively), and the association of both remained significant when bleeding severity score was adjusted for age (p=0.012 and p = 0.010, respectively). These associations persisted even if menorrhagia, the only gender-associated bleeding category, was excluded from the analysis. This is the first report of a statistically significant association of ITGA2 haplotype -52T with risk for bleeding in vivo, a finding that is consistent with the significant attenuation of ITGA2 transcription by this promoter haplotype in in vitro studies. No statistically significant association was observed with the major haplotypes of six other candidate genes, GP1BA, ITGB3, GP6, VWF, FGB, and IL6. Increased plasma VWF:Ag levels were associated with VWF haplotype 1 (−1793G) (p=0.04), although the impact of this VWF haplotype 1 association was not of itself sufficiently strong to directly influence bleeding severity score.These results establish that genetic differences in the expression or activity of integrin receptor subunits alpha-2 and alpha-IIb can influence the bleeding phenotype of VWD type 2. Together with the results of our previous studies, these findings demonstrate that platelet receptor gene haplotypes can influence bleeding tendency not only in VWD type 1 but also in VWD type 2. Interestingly, the influence of GP6 haplotype b (Pro219) on bleeding seen in VWD type 1 was not evident in this study of VWD type 2 pedigrees. Further studies on additional VWD type 1 and type 2 pedigrees, particularly pedigrees with diverse racial and/or ethnic backgrounds, will enable us to distinguish the variety and extent of genetic differences that can influence bleeding tendency in this disease.

VON WILLEBRAND DISEASE: DIAGNOSIS AND TREATMENT

Background. Von Willebrand disease (VWD) is a most frequently inborn bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (VWF). VWF promotes platelet-vessel wall (adhesion) and plateletplatelet interaction (aggregation). It is also the carrier for factor VIII (F VIII) in plasma. A deficiency of VWF may results in impairment of both, primary and secondary haemostasis. Therefore, patients with VWD can have bleeding symptoms typical fot the defect in primary haemostasis (mucocutaneous haemorrhages). In severe deficiency of VWF there are also haemarthroses and haematomas typical for patients with coagulation defects. Several types and subtypes of VWD have been described. The diagnosis is based on measurements of VWF concentration and activity and F VIII activity in plasma. The tests identifying VWD subtypes are ristocetin induced platelet agglutination (RIPA), multimeric analysis of VWF and measurement of the binding of VWF to F VIII. Conclusions. Due to heterogeneity of VWF defects, the correct diagnosis of types and subtypes is sometimes difficult but is important for appropriate treatment. There are two main therapeutic options for patients with VWD: desmopressin and blood derived concentrates of F VIII/VWF. In certain cases antifibrinolytics and hormones can be suitable treatment. Desmopressin is the treatment of choice in patients with type 1 VWD. It raises endogenous F VIII and VWF and thereby corrects the intrinsic coagulation defect as well as the prolonged bleeding time (BT) or closure time (CT-PFA100) in most type 1 VWD patients. In type 3 and in the majority of type 2 patients desmopressin is not effective and it is necessary to use concentrates containing F VIII and VWF. These are always effective in raising of F VIII activity, whereas the BT/CT may not be completely corrected, but the normalisation of the BT/CT is not always necessary.

A Novel Monoclonal Antibody against the Extracellular Domain of GPIbβ Modulates vWF Mediated Platelet Adhesion

GPIbbeta is disulfide-linked to GPIbalpha to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbbeta is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbbeta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbbeta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbbeta, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbbeta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbbeta.

A Novel Tetrameric Venom Protein, Agglucetin from Agkistrodon acutus , Acts as a Glycoprotein Ib Agonist

A novel platelet agglutination inducer, agglucetin, was purified from the Formosan Agkistrodon acutus snake venom. It migrated as a single band with an apparent molecular mass of 58.8 kDa and two distinct bands of 16.2/14.5 kDa under non-reducing and reducing conditions by SDS-PAGE, respectively. Further confirmed by FPLC, electrospray ionization mass spectrometry and 2D-PAGE, native agglucetin exists as a tetramer composed of disulfide-linked alpha1, alpha2, beta1 and beta2 subunits. Partial N-terminal sequence of agglucetin subunit showed a high degree of homology to those of C-type lectin-like glycoprotein (GP) Ib binding proteins. Functional studies showed that agglucetin. in the absence of von Willebrand factor (vWF), dose-dependently induced platelet agglutination and caused a negligible elevation of intracellular Ca+2 mobilization and thromboxane B, formation in human platelet suspensions. Anti-GP Ib monoclonal antibodies (mAbs), AP1 or LJ-Ib1, specifically inhibited agglucetin-induced platelet agglutination in a dose-dependent manner. However, EDTA, arietin (a long chain RGD-containing disintegrin), 7E3 (an anti-GP IIb/IIIa mAb), heparin, hirudin, PGE1, or indomethacin exhibited no inhibitory effect on agglucetin-induced platelet agglutination. Furthermore, flow cytometric analysis revealed that FITC-agglucetin dose-dependently bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by anti-GP Ib mAb. It is concluded that agglucetin, acts specifically on an epitope of platelet membrane GP Ib overlapping with that of API, causing platelet agglutination in a Ca+2- and GP IIb/IIIa-independent manner.