VWF-mediated Platelet Aggregation Research Articles

In vitro assessment and phase I randomized clinical trial\xa0of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIb\u03b1

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

The von Willebrand factor Tyr2561 allele is a gain-of-function variant and a risk factor for early myocardial infarction

AbstractThe frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI), and 417 control patients (Ludwigshafen Risk and Cardiovascular Health Study) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele aged 55 years or younger revealed an elevated risk for repeated MI (odds ratio, 2.53; 95% confidence interval [CI], 1.07-5.98). The odds ratio was even higher in females aged 55 years or younger, at a value of 5.93 (95% CI, 1.12-31.24). Cone and plate aggregometry showed that compared with Phe2561, Tyr2561 was associated with increased platelet aggregate size both in probands' blood and with the recombinant variants. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared with Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF as a result of altered association of the C domains that disrupts the normal dimer interface. In summary, our data emphasize the functional effect of the VWF C4 domain for VWF-mediated platelet aggregation in a shear-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism.

The Von Willebrand Factor Tyr2561 Allele Is a Gain-of-Function Variant and a Potential Risk Factor for Early Myocardial Infarction

The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI) and 417 controls (Ludwigshafen Risk and Cardiovascular Health Study (LURIC)) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele ≤55 years revealed an elevated risk for repeated MI (Odds ratio: 2.53, 95% CI:1.07-5.98). The Odds ratio was even higher in females ≤55 years at a value of 5.93 (95% CI:1.12-31.24). Cone and plate aggregometry showed, that compared to Phe2561, Tyr2561 was associated with increased platelet aggregate size, both in probands' blood and with the recombinant variants. Inhibition of the VWF-GPIIb/IIIa interaction abrogated the effect. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared to Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF, as a result of altered association of the C-domains that disrupts the normal dimer interface. In summary, our data emphasize the functional impact of the VWF C4-domain for VWF-mediated platelet aggregation in a GPIIb/IIIa-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism. DisclosuresSchneppenheim:SHIRE: Consultancy; CSL Behring: Consultancy.

Results of the Randomized, Double-Blind, Placebo-Controlled, Phase 3 Hercules Study of Caplacizumab in Patients with Acquired Thrombotic Thrombocytopenic Purpura

Introduction: Acquired or immune-mediated thrombotic thrombocytopenic purpura (aTTP) is a life-threatening thrombotic microangiopathy characterized by severe thrombocytopenia, microangiopathic hemolytic anemia, and organ ischemia. Inhibitory autoantibodies cause a severe deficiency of the von Willebrand factor (vWF) cleaving enzyme ADAMTS13, leading to intravascular vWF-platelet aggregation and microvascular thrombosis. The mainstays of treatment are plasma exchange (PE) and immunosuppression. Caplacizumab, a bivalent Nanobody, targets the A1 domain of vWF, inhibiting the interaction between ultra-large vWF and platelets. Methods: Patients with an acute episode of aTTP who had received one PE treatment were randomized 1:1 to placebo or 10 mg caplacizumab, in addition to daily PE and corticosteroids. A single IV dose of study drug was given before the first on-study PE and a SC dose was given daily during the PE period and 30 days thereafter. If at the end of this period there was evidence of ongoing disease, such as suppressed ADAMTS13 activity, investigators were encouraged to extend the blinded treatment for a maximum of 4 weeks together with optimization of immunosuppression. All patients entered a 28-day treatment-free follow up period after the last dose of study drug (Figure 1). Primary endpoint was time to platelet count response, defined as platelet count ≥ 150×109/L with stop of daily PE within 5 days. There were 4 key secondary endpoints, hierarchically ranked. The 1st was a composite of aTTP-related death, aTTP recurrence, or major thromboembolic event during the study drug treatment period. A blinded, independent committee adjudicated aTTP-related deaths and major thromboembolic events. The 2nd looked at recurrences during the entire study period, including the follow up period. The 3d evaluated refractoriness to therapy, defined as absence of platelet count doubling after 4 days of treatment and LDH still above normal. The 4th was the time to normalization of 3 organ damage markers: LDH, cardiac troponin I and serum creatinine. Results: 145 patients were randomized, 73 to placebo and 72 to caplacizumab. Demographics and baseline disease characteristics were balanced between groups, except for a higher proportion of initial episodes in the caplacizumab arm. Compared to patients treated with placebo, those on caplacizumab were >50% more likely to achieve a platelet response at any given time point (platelet count normalization rate 1.55, 95% CI 1.10 - 2.20, p Conclusions: Results of the Phase 3 HERCULES study confirm that treatment with caplacizumab reduces the time to platelet count response, resulting in faster resolution of aTTP. Treatment with caplacizumab also resulted in a highly clinically meaningful reduction in aTTP-related death, recurrence of aTTP, or a major thromboembolic event during study drug treatment. The relapses after stop of study drug in patients with ADAMTS13 activity Disclosures Scully:Ablynx: Honoraria, Research Funding; Shire: Honoraria, Research Funding; Novartis: Honoraria; Alexion: Honoraria. Cataland:Ablynx NV: Membership on an entity9s Board of Directors or advisory committees. Peyvandi:Ablynx, Roche: Membership on an entity9s Board of Directors or advisory committees; Ablynx, Bayer, Grifols, Novo Nordisk, Sobi: Speakers Bureau;Freeline, Kedrion, LFB, Octapharma: Consultancy. Coppo:Ablynx: Membership on an entity9s Board of Directors or advisory committees; Alexion: Membership on an entity9s Board of Directors or advisory committees; Octapharma: Membership on an entity9s Board of Directors or advisory committees; Shire: Membership on an entity9s Board of Directors or advisory committees. Knobl:Novo Nordisk: Consultancy, Research Funding; Shire: Consultancy, Research Funding. Kremer Hovinga:Baxalta/Shire: Other: unrestricted grant hereditary TTP registry; Ablynx NV: Membership on an entity9s Board of Directors or advisory committees. Metjian:Ablynx NV, Shire, Omeros: Research Funding; Shire: Membership on an entity9s Board of Directors or advisory committees. de la Rubia:Amgen: Other: Honoraria; Celgene: Other: Honoraria; Janssen: Other: Honoraria. Pavenski:Alexion Pharmaceuticals: Honoraria; Novartis: Honoraria; Ablynx: Other: participation in industry sponsored RCT; CSL Behring: Research Funding. Callewaert:Ablynx NV: Employment. Biswas:Ablynx NV: Employment. De Winter:Ablynx NV: Employment. Zeldin:Ablynx NV: Employment.

A biophysical view on von Willebrand factor activation.

The process of hemostatic plug formation at sites of vascular injury crucially relies on the large multimeric plasma glycoprotein von Willebrand factor (VWF) and its ability to recruit platelets to the damaged vessel wall via interaction of its A1 domain with platelet GPIbα. Under normal blood flow conditions, VWF multimers exhibit a very low binding affinity for platelets. Only when subjected to increased hydrodynamic forces, which primarily occur in connection with vascular injury, VWF can efficiently bind to platelets. This force-regulation of VWF's hemostatic activity is not only highly intriguing from a biophysical perspective, but also of eminent physiological importance. On the one hand, it prevents undesired activity of VWF in intact vessels that could lead to thromboembolic complications and on the other hand, it enables efficient VWF-mediated platelet aggregation exactly where needed. Here, we review recent studies that mainly employed biophysical approaches in order to elucidate the molecular mechanisms underlying the complex mechano-regulation of the VWF-GPIbα interaction. Their results led to two main hypotheses: first, intramolecular shielding of the A1 domain is lifted upon force-induced elongation of VWF; second, force-induced conformational changes of A1 convert it from a low-affinity to a high-affinity state. We critically discuss these hypotheses and aim at bridging the gap between the large-scale behavior of VWF as a linear polymer in hydrodynamic flow and the detailed properties of the A1-GPIbα bond at the single-molecule level.

Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism

The large multimeric glycoprotein von Willebrand Factor (VWF) plays a pivotal adhesive role during primary hemostasis. VWF is cleaved by the protease ADAMTS13 as a down-regulatory mechanism to prevent excessive VWF-mediated platelet aggregation. For each VWF monomer, the ADAMTS13 cleavage site is located deeply buried inside the VWF A2 domain. External forces in vivo or denaturants in vitro trigger the unfolding of this domain, thereby leaving the cleavage site solvent-exposed and ready for cleavage. Mutations in the VWF A2 domain, facilitating the cleavage process, cause a distinct form of von Willebrand disease (VWD), VWD type 2A. In particular, the VWD type 2A Gly1629Glu mutation drastically accelerates the proteolytic cleavage activity, even in the absence of forces or denaturants. However, the effect of this mutation has not yet been quantified, in terms of kinetics or thermodynamics, nor has the underlying molecular mechanism been revealed. In this study, we addressed these questions by using fluorescence correlation spectroscopy, molecular dynamics simulations, and free energy calculations. The measured enzyme kinetics revealed a 20-fold increase in the cleavage rate for the Gly1629Glu mutant compared with the wild-type VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites of the VWF multimer at the same time. According to our simulations and free energy calculations, the Gly1629Glu mutation causes structural perturbation in the A2 domain and thereby destabilizes the domain by ∼10 kJ/mol, promoting its unfolding. Taken together, the enhanced proteolytic activity of Gly1629Glu can be readily explained by an increased availability of the ADAMTS13 cleavage site through A2-domain-fold thermodynamic destabilization. Our study puts forward the Gly1629Glu mutant as a very efficient enzyme substrate for ADAMTS13 activity assays.

Risk Factors Associated with Multiple End-Organ Damage in Idiopathic Thrombotic Thrombocytopenic Purpura (TTP) Patients

Introduction: Idiopathic thrombotic thrombocytopenic purpura (TTP) is a rare hematological disorder caused by deficiency of ADAMTS13 activity and subsequent development of vWF-platelets-rich microthrombi leading to tissue ischemia and organ dysfunction. Standard treatment consists of daily plasma exchange (PEX) to remove the autoantibodies and replenishes ADAMTS13 activity and immunosuppressive therapy for patients with autoimmune process. Recent publication showed that caplacizumab, an anti-vWF humanized nanobody which inhibits the interaction between vWF multimers and platelets, can be used together with PEX to reduce time to platelet normalization but not risk of relapse when compared to placebo. However, incorporate caplacizumab with PEX in high risk TTP patients maybe even more important to reduce long-term organ damage. Therefore, we performed a retrospective single center analysis of TTP patients to identify the incidence and risk factors associated with end-organ damage. Method: Sixteen adult patients with idiopathic TTP were identified from January 2005 through December 2015. Idiopathic TTP was defined as thrombotic microangiopathy with an ADAMTS13 ≤10%. Cardiac involvement was defined as an increased Troponin I (TnI) level >0.1µg L-1. Renal involvement was defined as serum creatinine >1.30mg/dL. Of note, all the 16 patients had normal baseline renal function. Neurological involvement was defined as documentation of confusion, headaches, seizure, aphasia and syncope. The time to a platelet response was defined as a platelet count that was ≥150,000/mm3 for at least 48 hours. All patients were treated with PEX, corticosteroids, ± rituximab. Results: The mean (range) age was 48 (23 to 78) and the female: male ratio was 12:4. All (16) patients had end-organ involvements: 16 (100%) neurological, 6 (37.5%) renal, and 4 (25%) cardiac. Seven patients (43.75%) had single organ involvement (Group one), 9 patients (56.25%) had two or three organ involvement (Group two). The mean age for Group one was 44 as compared to 51 for Group two. The mean LDH level on admission was 987 IU/L (range: 417-1,289 IU/L) for Group one as compared to 1,715 IU/L (range: 336-4,455 IU/L) for Group two (p value: 0.06). The mean platelet count on admission was 20 k/µL for Group one as compared to 18 k/µL for Group two. The mean hemoglobin level on admission was 7.5g/dL (range: 5.3-8.6g/dL) for Group one as compared to 8.78g/dL (range: 8.1-10.5g/dL) for Group two. The median time to a platelet response was 5 days in this study population. Conclusions: Our study showed that more than 50% of TTP patients presented with multi-organ involvement. Very high LDH at initial presentation prior to PEX was identified as a probable single risk factor associated with multi-organ involvement. Prompt arrest of vWF-mediated platelet aggregation with agents such as calpalacizumab in TTP patients could prevent or minimize early irreversible end-organ damage due to tissue ischemia. Identifying risk factors for severe end-organ involvement will be important to study the potential benefit of incorporating calpalacizumab into TTP treatment. Further study on the risk factors for end-organ involvements in TTP patients and their long-term outcome at a multi-center level and with a larger patient population is warranted. Disclosures No relevant conflicts of interest to declare.

Heat-shock protein gp96/grp94 is an essential chaperone for the platelet glycoprotein Ib-IX-V complex

The platelet glycoprotein Ib-IX-V complex (GPIb-IX-IV) is the receptor for VWF and is responsible for VWF-mediated platelet activation and aggregation. Loss of the GPIb-IX-V complex is pathogenic for Bernard-soulier Syndrome (BSS), which is characterized by macrothrombocytopenia and impaired platelet function. It remains unclear how the GPIb-IX-V complex is assembled and whether there is a role for a specific molecular chaperone in the process. In the present study, we report that the assembly of the GPIb-IX-V complex depends critically on a molecular chaperone in the endoplasmic reticulum (ER): gp96 (also known as grp94 and HSP90b1). gp96/grp94 deletion in the murine hematopoietic system results in thrombocytopenia, prolonged bleeding time, and giant platelets that are clinically indistinguishable from human BSS. Loss of gp96/grp94 in vivo and in vitro leads to the concomitant reduction in GPIb-IX complex expression due to ER-associated degradation. We further demonstrate that gp96/grp94 binds selectively to the GPIX subunit, but not to gpIbα or gpIbβ. Therefore, we identify the platelet GPIX subunit of the GPIb-IX-V complex as an obligate and novel client of gp96/grp94.

Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of Anti-Vwf Nanobody® ALX-0681 After Single and Multiple Subcutaneous Administrations to Healthy Volunteers.

Abstract 1063Poster Board I-85ALX-0681 is a humanized bivalent Nanobody®, that binds to the A1 domain of von Willebrand factor (vWF) and hence blocks its interaction with platelet receptor GPIb-IX-V. Given its mode of action, ALX-0681 could provide an alternative treatment option for thrombotic thrombocytopenic purpura (TTP), a rare and life-threatening condition characterized by systemic platelet aggregation in the microcirculation mediated by activated vWF multimers. The goal of this Phase I trial in healthy volunteers was to determine the maximum tolerated dose (MTD) or biologically effective dose (BED) and the Phase II dosing and scheduling of ALX-0681, in order to support the further clinical development of ALX-0681 in TTP patients. In total, 36 healthy volunteers were included in this randomized, placebo-controlled study to evaluate the safety of single ascending doses and multiple doses of ALX-0681 administered subcutaneously (s.c.) (Table 1).Table 1Dosing schedule for Phase I trial with ALX-0681CohortDose (mg)Number of daily dosesSubjects receiving ALX-0681Subjects receiving placeboSingle doseCohort 12131Cohort 24131Cohort 38131Cohort 416131Cohort 510131Multiple doseCohort 610762Cohort 7101462Study endpoints included safety (dose limiting toxicities, adverse events (AEs) and immunogenicity), pharmacokinetics (PK), pharmacodynamics (PD) and pharmacological efficacy of ALX-0681. The latter endpoint was addressed by measuring the ristocetin cofactor (RICO) biomarker, reflecting vWF mediated inhibition of platelet aggregation. ALX-0681 was safe and well tolerated at all dose levels (Table 2). One unrelated SAE (meniscus lesion) occurred. The number of observed signs of bleeding and bruises increased with increasing treatment duration. However, all these events were of mild intensity. No signs of immunogenicity were observed for a minimum of 45 days after the last injection.Table 2Summary of main safety results (number (%) of subjects with event)Dose levelSubjects (n)AESAEBleedingHematoma at injection siteHematoma at blood sampling siteOther hematomaSingle dose2 mg32 (67)0 (0)1 (33)0 (0)1 (33)0 (0)4 mg32 (67)0 (0)1 (33)0 (0)0 (0)0 (0)8 mg33 (100)0 (0)0 (0)0 (0)1 (33)0 (0)16 mg33 (100)0 (0)0 (0)0 (0)0 (0)1 (33)10 mg31 (33)0 (0)0 (0)0 (0)1 (33)0 (0)Placebo53 (60)0 (0)0 (0)0 (0)0 (0)0 (0)Multiple dose10 mg (7d)66 (100)1 (17)5 (83)1 (17)0 (0)3 (50)Placebo (7d)22 (100)0 (0)1 (50)0 (0)1 (50)0 (0)10 mg (14d)66 (100)0 (0)5 (83)5 (83)4 (67)5 (83)Placebo (14d)22 (100)0 (0)1 (50)0 (0)0 (0)0 (0)PK analysis showed a rapid increase in ALX-0681 plasma concentration (tmax = 4-10 h post dose), followed by a slow elimination phase (t1/2 = 10-78 h). All subjects dosed with ALX-0681 at 8 mg or higher showed complete inhibition of RICO activity to < 20% with an onset of 1-6 h post dose. This inhibition was maintained until 12-360 h post dose, depending on the dose level (Table 3). Overall, 20 (74%) and 17 (63%) of ALX-0681 treated subjects experienced a drop in vWF and FVIII levels below 50% of pre-dose levels, respectively. These events were all transient and not clinically significant.Table 3Summary of main PD results (number (%) of subjects with event)Dose levelSubjects (n)RICO < 20%vWF < 50%FVIII < 50%Subjects (%)Start (h)*Stop (h)*Single dose2 mg32 (67)2-412-183 (100)0 (0)4 mg32 (67)4-618-361 (33)1 (33)8 mg33 (100)2-418-483 (100)3 (100)16 mg33 (100)1-4480 (0)2 (67)10 mg33 (100)2-624-363 (100)3 (100)Placebo50 (0)NANA0 (0)0 (0)Multiple dose10 mg (7d)66 (100)2-4168-1925 (83)3 (50)Placebo (7d)20 (0)NANA0 (0)0 (0)10 mg (14d)66 (100)2-4336-3605 (83)5 (83)Placebo (14d)20 (0)NANA0 (0)0 (0)*Time relative to first administration NA: not applicable In conclusion, ALX-0681 administered s.c. for up to 14 days was well tolerated and did not result in any clinically significant AEs. No local reactions, local intolerances or signs of clinically relevant bleeding were reported. The PD marker indicated complete inhibition of vWF mediated platelet aggregation following single daily s.c. injections of 10 mg, which was maintained over the 2 weeks treatment period. Multiple daily administration of s.c. injections of ALX-0681 did not result in an immunogenic reaction for a minimum of 45 days following completion of treatment. Based on the results of this study, ALX-0681 development will be advanced into a Phase II study in TTP patients to investigate the safety and efficacy of ALX-0681 in the target patient population. Disclosures:Abd-Elaziz:Ablynx NV: Consultancy. Kamphuisen:Ablynx NV: Consultancy. Lyssens:Ablynx NV: Employment. Reuvers:Ablynx NV: Consultancy. den Daas:Ablynx NV: Consultancy. Van Bockstaele:Ablynx NV: Employment. Vercruysse:Ablynx NV: Employment. Ulrichts:Ablynx NV: Employment. Baumeister:Ablynx NV: Employment. Crabbe:Ablynx NV: Employment. Compernolle:Ablynx NV: Employment. Holz:Ablynx NV: Employment.

Identification of Peptide Antagonists to Glycoprotein Ibα That Selectively Inhibit von Willebrand Factor Dependent Platelet Aggregation

GPIbalpha is an integral membrane protein of the GPIb-IX-V complex found on the platelet surface that interacts with the A1 domain of von Willebrand factor (vWF-A1). The interaction of GPIbalpha with vWF-A1 under conditions of high shear stress is the first step in platelet-driven thrombus formation. Phage display was used to identify peptide antagonists of the GPIbalpha-vWF-A1 interaction. Two nine amino acid cysteine-constrained phage display libraries were screened against GPIbalpha revealing peptides that formed a consensus sequence. A peptide with sequence most representative of the consensus, designated PS-4, was used as the basis for an optimized library. The optimized selection identified additional GPIbalpha binding peptides with sequences nearly identical to the parent peptide. Surface plasmon resonance of the PS-4 parent and two optimized synthetic peptides, OS-1 and OS-2, determined their equilibrium dissociation GPIbalpha binding constants ( K Ds) of 64, 0.74, and 31 nM, respectively. Isothermal calorimetry corroborated the K D of peptide PS-4 with a resulting affinity value of 68 nM. An ELISA demonstrated that peptides PS-4, OS-1, and OS-2 competitively inhibited the interaction between the vWF-A1 domain and GPIbalpha-Fc in a concentration-dependent manner. All three peptides inhibited GPIbalpha-vWF-mediated platelet aggregation induced under high shear conditions using the platelet function analyzer (PFA-100) with full blockade observed at 150 nM for OS-1. In addition, OS-1 blocked ristocetin-induced platelet agglutination of human platelets in plasma with no influence on platelet aggregation induced by several agonists of alternative platelet aggregation pathways, demonstrating that this peptide specifically disrupted the GPIbalpha-vWF-A1 interaction.

A highly-sensitive plasma von Willebrand factor ristocetin cofactor (VWF:RCo) activity assay by flow cytometry

Assays of plasma von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) are essential for the laboratory diagnosis of von Willebrand disease (VWD) and for monitoring therapy. However, current manual or automated VWF:RCo assay methods have relatively poor operating characteristics. Our goal was to develop and validate a simple, accurate, specific and sensitive platelet-based VWF:RCo assay. Using green or red fluorochrome-labeled, fixed normal platelets and normal or patient plasma, ristocetin-dependent and VWF-mediated platelet aggregation was detected by flow cytometry. VWF:RCo activity was assayed as the number of double-positive events (green and red) among all green or red events, relative to the calibrator plasma signal (6-150% or IU dL(-1)), and reported as percent or IU dL(-1). We tested plasma samples from normal donors (n = 51) and known VWD patients (type 1, n = 16; type 2, n = 17) based on clinical history, levels of plasma VWF antigen (VWF:Ag), VWF:RCo activity (manual platelet aggregometry/agglutination assay), factor (F) VIII activity and VWF multimer analysis. For normal donors and type 1 VWD patients, VWF:RCo activity by flow cytometry vs. manual platelet aggregation correlated closely (R2 = 0.74), and VWF:RCo/VWF:Ag ratios did not differ significantly. In contrast, VWF:RCo/VWF:Ag ratios for type 2 VWD subtypes were significantly lower using VWF:RCo by flow cytometry vs. manual platelet aggregation assay (P < 0.01), especially for type 2A VWD patients. This new flow cytometry-based VWF:RCo assay is simple, accurate, specific and sensitive, particularly for type 2 VWD.

Antidote-Controlled Platelet Inhibition Targeting von Willebrand Factor with Aptamers

Thrombus formation is initiated by platelets and leads to cardiovascular, cerebrovascular, and peripheral vascular disease, the leading causes of morbidity and mortality in the Western world. A number of antiplatelet drugs have improved clinical outcomes for thrombosis patients. However, their expanded use, especially in surgery, is limited by hemorrhage. Here, we describe an antiplatelet agent that can have its activity controlled by a matched antidote. We demonstrate that an RNA aptamer targeting von Willebrand factor (VWF) can potently inhibit VWF-mediated platelet adhesion and aggregation. By targeting this important adhesion step, we show that the aptamer molecule can inhibit platelet aggregation in PFA-100 and ristocetin-induced platelet aggregation assays. Furthermore, we show that a rationally designed antidote molecule can reverse the effects of the aptamer molecule, restoring platelet function quickly and effectively over a clinically relevant period. This aptamer-antidote pair represents a reversible antiplatelet agent inhibiting a platelet specific pathway. Furthermore, it is an important step towards creating safer drugs in clinics through the utilization of an antidote molecule.

Why Do We Need ADAMTS13?

ADAMTS13 is a member of a newly recognized zinc metalloprotease family that has been the subject of intense interest in recent years. Cleavage of von Willebrand factor by this protease is essential for preventing the development of thrombotic thrombocytopenic purpura (TTP) Von Willebrand factor (VWF), synthesized in vascular endothelial cells and megakaryocytes, supports platelet adhesion and aggregation at sites of vessel injury under high shear stress conditions. VWF exists in the circulation as a series of multimers, which were once believed to be released directly from vascular endothelial cells. By late 1980’s, much was known about the physiology and genetics of VWF. Nevertheless, three characteristics of VWF-platelet interaction in hemostasis had resisted efforts to unravel their molecular mechanisms (Table 1). First, VWF-platelet binding occurs only at sites of vessel injury but not in the circulation. There are two receptors on platelet surface that are engaged in VWF binding: glycoproteins αIIbβ3 and Ib/IX/V. Much is known about the regulation of VWF–αIIbβ3: αIIbβ3, normally in an inactive conformation, is transformed upon platelet activation to a competent form for ligand binding. In contrast, the regulation of VWF–Ib/IX/V remained mostly unclear. There is no definitive evidence that Ib/IX/V requires activation. What processes prevent VWF-platelet binding in the circulation but allows it to occur at sites of vessel injury? Second, several lines of evidence suggest that the large VWF multimers arc hemostatically more effective than small multimers. What is the molecular basis of this size dependence? Third, in experimental models, high levels of shear stress enhance the VWF-mediated platelet aggregation. What are the molecular features of VWF that enable VWF to become more effective under high shear stress conditions? Table 1 Characteristics of VWF-platelel interaction and their causes

The Cytoplasmic Tail of GPIbß Modulates Signalling Induced by GPIb/VWF Interactions under High Shear Conditions.

The platelet glycoprotein (GP) Ib/IX/V receptor complex controls crucial steps in haemostasis and thrombosis by mediating adhesion to von Willebrand factor (VWF). Nevertheless, the downstream consequences of VWF binding to GPIb remain unclear. The GPIb/IX/V complex consists of four polypeptides GPIbα, GPIbβ, GPIX and GPV. We have shown that a palmitylated (Pal-) peptide based on the sequence of the GPIbβ cytoplasmic domain between R151 and A161 (Pal-RRLRARARARA abbreviated to RARA) increases the velocity of platelets rolling on VWF, and inhibits VWF-dependent adhesion and aggregation.The peptide RARA is delivered into platelets and does not cause any reduction in the surface expression of GPIb-IX-V. We examined the effect of RARA and control peptides (Pal-RAAAARARARA and Pal-RRLRADADADA) on GPIb/IX/V-mediated tyrosine kinase signalling induced by VWF/GPIb interactions under shear conditions using a cone and plate viscometer. Whole platelet lysate phosphotyrosine immunoblots were characterised in the presence and absence of RARA and the control peptides before and after shearing at 120 dynes/cm2 for 5 mins. We then further investigated the effect of RARA on PI-3 kinase signalling, the tyrosine kinase Src and calcium influx under the conditions described above.The platelet permeable peptide RARA alone specifically caused protein tyrosine phosphorylation under resting conditions and caused platelet microaggregate formation with a 35% reduction in single platelets as assessed by flow cytometry. In addition RARA significantly inhibited shear-induced aggregation. When tyrosine phosphorylation was examined in platelets sheared for 5 minutes at 120 dynes/cm2, we observed RARA failed to inhibit tyrosine phosphorylation despite inhibiting shear-induced aggregation.RARA caused Src kinase activation as determined by site-specific phosphorylation of Y416 on Src. Furthermore the Src inhibitor PP1 inhibited RARA induced tyrosine phosphorylation. RARA caused a Src-dependent increase in intracellular [Ca2+] and inhibited any further increase in intracellular calcium induced by thrombin. Pretreatment of platelets with the PI-3 kinase inhibitor wortmannin demonstrated that tyrosine phosphorylation induced by RARA was not PI-3 kinase dependent.These results indicate that a platelet permeable peptide corresponding to specific residues of the cytoplasmic tail of GPIbβ directly stimulates Src, and suggest that the mechanism of its inhibitory effect on VWF-mediated platelet adhesion and aggregation involves the tyrosine kinase-mediated desensitisation of a GPIb/IX/V-triggered signalling pathway. This study demonstrates a novel role for the GPIbβ cytoplasmic tail in mediating Src-dependent shear induced signalling through GPIb/VWF.

Effects of ticlopidine on von Willebrand factor-mediated shear-induced platelet activation and aggregation

Ticlopidine, recently classified as a P2Y 12 ADP receptor blockade, is known to be an effective antiplatelet agent preventing arterial thrombosis, e.g., myocardial infarction or cerebral infarction, but the mechanism of the in vivo antithrombotic effects of ticlopidine is not fully understood. Blood was drawn from seven normal volunteers before and 7 days after consecutive intake of ticlopidine, and 1 day after oral intake of aspirin after 7 days of ticlopidine wash-out period. Effects of drug intake on shear-induced von Willebrand factor (vWF) binding to platelets, platelet activation evidenced by P-selectin surface expression and translocation of GP Ib f , and vWF-mediated platelet aggregation, were investigated by using an optically modified cone-plate viscometer and quantitative flow cytometry. The maximum extent of platelet aggregation occurring under a high shear rate of 10800 s -1 , presumably mediated by vWF, was not significantly influenced by either ticlopidine or aspirin. However, significant dissociation of once aggregated platelets occurred in samples obtained after ticlopidine intake (25.4 - 9.3%, P = 0.00030) and less significantly after aspirin intake (15.9 - 5.7%, P = 0.0013), while only insignificant and modest dissociation occurred in controls (6.3 - 4.4%, n.s.). Binding experiments also revealed that the shear-induced vWF binding to platelets was significantly inhibited by ticlopidine, and less significantly by aspirin, although other indicators of platelet activation, such as shear-induced P-selectin expression and GP Ib f translocation were not influenced by either ticlopidine or aspirin. We demonstrate here that platelet aggregation mediated by von Willebrand factor (vWF) under a high shear rate of 10800 s -1 cannot be stabilized and that dissociation occurs readily when ADP receptor stimulation is blocked by continuous intake of ticlopidine, indicating that these effects on platelet thrombus formation may contribute to the in vivo antithrombotic effects of ticlopidine.

M r 6,400 aurin tricarboxylic acid directly activates platelets

ATA is a novel anticoagulant polymetic anionic aromatic compound that inhibits von Willebrand factor binding to platelet glycoprotein Ib and thereby prevents ristocetin- and shear stress-induced platelet aggregation. To investigate its mechanism of action, ATA fractions of homogeneous M r have been prepared by size exclusion chromatography. ATA fractions of M r ≥ 2,500 are most effective at inhibiting vWF-mediated platelet aggregation, and ATA of M r = 2,500 also inhibits thrombin-induced platelet activation. Paradoxical results were observed in studies of ATA with M r = 6,400. This fraction of ATA stimulates aggregation of washed platelets or platelet-rich plasma. The dose/response of aggregation shows a bell-shaped curve with maximal aggregation at approximately 2 μg/ml. Platelet aggregation is associated with phosphoinositide turnover and protein kinase C- and calcium-dependent protein phosphorylation. Platelet signalling responses to ATA are inhibited by platelet pretreatment with PGI 2 or dibutyryl-cyclic AMP, but are unaffected by inhibiting platelet cyclooxygenase with aspirin. These results suggest that M r 6,400 ATA directly activates platelet phospholipase C to initiate platelet aggregation. This effect, unique to M r 6,400 ATA, could potentially mitigate ATA's beneficial anti-thrombotic effect on vWF-mediated platelet responses, and should be considered when analyzing results of experiments that utilize unfractionated ATA.

Isolation from commercial aurintricarboxylic acid of the most effective polymeric inhibitors of von Willebrand factor interaction with platelet glycoprotein Ib. Comparison with other polyanionic and polyaromatic polymers

Solutions of commercial aurintricarboxylic acid (ATA) inhibit ristocetin- or shear stress-induced, von Willebrand factor (vWF)- mediated platelet aggregation by interacting with vWF and blocking its attachment to platelet membrane glycoprotein Ib. ATA has also been shown to prevent cyclic platelet clumping in a dog model of coronary artery thrombosis. Because these ATA solutions are actually a heterogeneous mixture of polyanionic, polycarboxylic polyaromatic polymers of molecular weight (Mr) 200 to greater than 6,000, we separated the most effective inhibitory components of commercial ATA using exclusion chromatography. ATA polymers larger than Mr 700 inhibited ristocetin-induced, vWF-mediated platelet aggregation more effectively than smaller ATA polymers, whereas shear-induced, vWF- mediated platelet aggregation was optimally inhibited by ATA polymers of Mr greater than or equal to 2,500. Platelet aggregation mediated by vWF was not inhibited by a nonphenolic, polyanionic polymer (polyglutamic acid) or by a polyphenolic ATA-like polymer (aurin) devoid of carboxyl groups. Polyanionic, polysulfonated aromatic polymers (polystyrene sulfonate) of Mr 35, 17.4, 8, and 4.6 x 10(3) inhibited ristocetin- and shear-induced, vWF-mediated aggregation with less potency on a mass/volume basis than large polymers of ATA. We conclude that a polyanionic, polycarboxylated, polyphenolic ATA polymer of Mr 2,500 is optimally potent as an inhibitor of shear- and ristocetin-induced, vWF-mediated platelet aggregation and is likely to be more effective than solutions of commercial ATA as an anti-arterial thrombotic agent.

Isolation From Commercial Aurintricarboxylic Acid of the Most Effective Polymeric Inhibitors of von Willebrand Factor Interaction With Platelet Glycoprotein Ib. Comparison With Other Polyanionic and Polyaromatic Polymers

Solutions of commercial aurintricarboxylic acid (ATA) inhibit ristocetin- or shear stress-induced, von Willebrand factor (vWF)-mediated platelet aggregation by interacting with vWF and blocking its attachment to platelet membrane glycoprotein Ib. ATA has also been shown to prevent cyclic platelet clumping in a dog model of coronary artery thrombosis. Because these ATA solutions are actually a heterogeneous mixture of polyanionic, polycarboxylic polyaromatic polymers of molecular weight (Mr) 200 to greater than 6,000, we separated the most effective inhibitory components of commercial ATA using exclusion chromatography. ATA polymers larger than Mr 700 inhibited ristocetin-induced, vWF-mediated platelet aggregation more effectively than smaller ATA polymers, whereas shear-induced, vWF-mediated platelet aggregation was optimally inhibited by ATA polymers of Mr greater than or equal to 2,500. Platelet aggregation mediated by vWF was not inhibited by a nonphenolic, polyanionic polymer (polyglutamic acid) or by a polyphenolic ATA-like polymer (aurin) devoid of carboxyl groups. Polyanionic, polysulfonated aromatic polymers (polystyrene sulfonate) of Mr 35, 17.4, 8, and 4.6 x 10(3) inhibited ristocetin- and shear-induced, vWF-mediated aggregation with less potency on a mass/volume basis than large polymers of ATA. We conclude that a polyanionic, polycarboxylated, polyphenolic ATA polymer of Mr 2,500 is optimally potent as an inhibitor of shear- and ristocetin-induced, vWF-mediated platelet aggregation and is likely to be more effective than solutions of commercial ATA as an anti-arterial thrombotic agent.

Aurin tricarboxylic acid: a novel inhibitor of the association of von Willebrand factor and platelets

Shear stress activated platelets undergo aggregation in the presence of large or unusually large von Willebrand factor (vWF) multimers without the addition of ristocetin or any other exogenous chemical. This phenomenon may be analogous to the platelet aggregation that leads to thrombosis in the narrowed arteries and arterioles of patients with atherosclerosis or vasospasm. A triphenyl-methyl compound, aurin tricarboxylic acid (ATA), inhibits shear-induced, vWF-mediated platelet aggregation in platelet-rich plasma (PRP) in concentrations above 200 mumol/L and in buffer suspensions of washed platelets at a concentration of 0.1 mumol/L. In a concentration-dependent manner, ATA also inhibits ristocetin-induced, vWF-mediated platelet clumping in both fresh and formaldehyde-fixed platelet suspensions. This inhibition can be overcome by increasing the concentration of vWF, following the kinetics of first order competitive inhibition. ATA prevents the attachment to platelets of the largest vWF multimeric forms found in normal plasma and of the unusually large vWF multimers derived from endothelial cells. The rate of aggregation and degree of inhibition by ATA is not accounted for by the binding of ristocetin or calcium. Arachidonic acid- and adenosine diphosphate (ADP)-induced aggregation are not inhibited by ATA. Platelets incubated with ATA can be easily separated from the compound. However, ATA binds to large vWF multimeric forms and inhibits their ristocetin-induced interaction with platelet glycoprotein Ib. Because ATA also inhibits shear-induced, vWF-mediated platelet aggregation in vitro in the absence of ristocetin, it may be a useful prototype compound to impede the development of arterial thrombosis in vivo.

Aurin Tricarboxylic Acid: A Novel Inhibitor of the Association of von Willebrand Factor and Platelets

Shear stress activated platelets undergo aggregation in the presence of large or unusually large von Willebrand factor (vWF) multimers without the addition of ristocetin or any other exogenous chemical. This phenomenon may be analogous to the platelet aggregation that leads to thrombosis in the narrowed arteries and arterioles of patients with atherosclerosis or vasospasm. A triphenyl-methyl compound, aurin tricarboxylic acid (ATA), inhibits shear-induced, vWF-mediated platelet aggregation in platelet-rich plasma (PRP) in concentrations above 200 mumol/L and in buffer suspensions of washed platelets at a concentration of 0.1 mumol/L. In a concentration-dependent manner, ATA also inhibits ristocetin-induced, vWF-mediated platelet clumping in both fresh and formaldehyde-fixed platelet suspensions. This inhibition can be overcome by increasing the concentration of vWF, following the kinetics of first order competitive inhibition. ATA prevents the attachment to platelets of the largest vWF multimeric forms found in normal plasma and of the unusually large vWF multimers derived from endothelial cells. The rate of aggregation and degree of inhibition by ATA is not accounted for by the binding of ristocetin or calcium. Arachidonic acid- and adenosine diphosphate (ADP)-induced aggregation are not inhibited by ATA. Platelets incubated with ATA can be easily separated from the compound. However, ATA binds to large vWF multimeric forms and inhibits their ristocetin-induced interaction with platelet glycoprotein Ib. Because ATA also inhibits shear-induced, vWF-mediated platelet aggregation in vitro in the absence of ristocetin, it may be a useful prototype compound to impede the development of arterial thrombosis in vivo.