Level of von Willebrand factor to assess the occurrence and prognosis of acute myocardial infarction.

BACKGROUND: To observe and compare the differences in von Willebrand factor antigen (vWF:Ag) levels between patients with acute myocardial infarction (AMI) and healthy residents, and to determine whether this measure can be used to evaluate the incidence of AMI and whether or not to undertake cardiac bypass surgery. METHODS: A retrospective analysis was conducted using the clinical data of 110 patients with acute cardiovascular disease without bypass (no bypass group), 351 patients with AMI and bypass (bypass group), and 60 healthy volunteers (healthy group) who underwent physical examination between July 2018 and May 2019 in Tianjin Chest Hospital. The plasma vWF:Ag was measured and the receiver operating characteristic (ROC) curve was utilized to critically assess its efficacy in determining the occurrence and prognosis of AMI, and the Chi-square test was used to evaluate the correlation between vWF:Ag and clinicopathological factors. RESULTS: The plasma vWF:Ag was 201% (139%, 250%) in the bypass group, 118% (107%, 134%) in the non-bypass group, and 95.5% (85.25%, 102.75%) in the control group, and the differences were statistically significant (P<0.05). The component status of the plasma vWF:Ag used in the bypass group was greater as compared to that of the normal group (P<0.05) and the non-bypass group (P<0.05). The area under the ROC curve of plasma vWF:Ag level was 0.797 (95% CI: 0.749-0.845). When the medical decision level of plasma vWF:Ag was set at 155.5%, the sensitivity and specificity of predicting AMI were 68.9% and 86.7%, respectively. The levels of plasma vWF:Ag in patients with AMI were correlated with hypertension, diabetes, age, and history of cerebral infarction (P<0.05). CONCLUSIONS: The plasma vWF level can predict the occurrence of AMI and provides guidance for cardiac bypass surgery.

A critical evaluation of caplacizumab for the treatment of acquired thrombotic thrombocytopenic purpura.

Introduction: Acquired thrombotic thrombocytopenic purpura (aTTP) is a thrombotic microangiopathy caused by inhibitory autoantibodies against ADAMTS13 protein. Until recently, the combination of plasma exchange (PEX) and immunosuppression has been the standard front-line treatment in this disorder. However, aTTP-related mortality, refractoriness, and relapse are still a matter of concern. Areas covered: The better understanding of the pathophysiological mechanisms of aTTP has allowed substantial improvements in the diagnosis and treatment of this disease. Recently, the novel anti-VWF nanobody caplacizumab has been approved for acute episodes of aTTP. Caplacizumab is capable to block the adhesion of platelets to VWF, therefore inhibiting microthrombi formation in the ADAMTS13-deficient circulation. In this review, the characteristics of caplacizumab together with the available data of its efficacy and safety in the clinical setting will be analyzed. Besides, the current scenario of aTTP treatment will be provided, including the role of other innovative drugs. Expert opinion: With no doubt, caplacizumab is going to change the way we treat aTTP. In combination with standard treatment, caplacizumab can help to significantly reduce aTTP-related mortality and morbidity and could spare potential long-term consequences by minimizing the risk of exacerbation.

von Willebrand Factor Predicts Mortality in ACS Patients Treated with Potent P2Y12 Antagonists and is Inhibited by Aptamer BT200 Ex Vivo.

BACKGROUND: von Willebrand factor (VWF) is crucial for arterial thrombosis and its plasma levels are increased in acute coronary syndromes (ACSs). The effects of conventional platelet inhibitors are compromised by elevated VWF under high shear rates. BT200 is a third-generation aptamer that binds and inhibits the A1 domain of human VWF. This article aims to study whether VWF is a predictor of mortality in ACS patients under potent P2Y12 blocker therapy and to examine the effects of a VWF inhibiting aptamer BT200 and its concentrations required to inhibit VWF in plasma samples of patients with ACS. METHODS: VWF activity was measured in 320 patients with ACS, and concentration effect curves of BT200 were established in plasma pools containing different VWF concentrations. RESULTS: Median VWF activity in patients was 170% (interquartile range % confidence interval [CI]: 85-255) and 44% of patients had elevated (> 180%) VWF activity. Plasma levels of VWF activity predicted 1-year (hazard ratio [HR]: 2.68; 95% CI: 1.14-6.31; p < 0.024) and long-term (HR: 2.59; 95% CI: 1.10-6.09) mortality despite treatment with potent platelet inhibitors (dual-antiplatelet therapy with aspirin and prasugrel or ticagrelor). Although half-maximal concentrations were 0.1 to 0.2 µg/mL irrespective of baseline VWF levels, increasing concentrations (0.42-2.13 µg/mL) of BT200 were needed to lower VWF activity to < 20% of normal in plasma pools containing increasing VWF activity (p < 0.001). CONCLUSION: VWF is a predictor of all-cause mortality in ACS patients under contemporary potent P2Y12 inhibitor therapy. BT200 effectively inhibited VWF activity in a target concentration-dependent manner.

The aptamer BT200 effectively inhibits von Willebrand factor (VWF) dependent platelet function after stimulated VWF release by desmopressin or endotoxin.

Von Willebrand factor (VWF) plays a major role in arterial thrombosis. Antiplatelet drugs induce only a moderate relative risk reduction after atherothrombosis, and their inhibitory effects are compromised under high shear rates when VWF levels are increased. Therefore, we investigated the ex vivo effects of a third-generation anti-VWF aptamer (BT200) before/after stimulated VWF release. We studied the concentration-effect curves BT200 had on VWF activity, platelet plug formation under high shear rates (PFA), and ristocetin-induced platelet aggregation (Multiplate) before and after desmopressin or endotoxin infusions in healthy volunteers. VWF levels increased > 2.5-fold after desmopressin or endotoxin infusion (p < 0.001) and both agents elevated circulating VWF activity. At baseline, 0.51 µg/ml BT200 reduced VWF activity to 20% of normal, but 2.5-fold higher BT200 levels were required after desmopressin administration (p < 0.001). Similarly, twofold higher BT200 concentrations were needed after endotoxin infusion compared to baseline (p < 0.011). BT200 levels of 0.49 µg/ml prolonged collagen-ADP closure times to > 300 s at baseline, whereas 1.35 µg/ml BT200 were needed 2 h after desmopressin infusion. Similarly, twofold higher BT200 concentrations were necessary to inhibit ristocetin induced aggregation after desmopressin infusion compared to baseline (p < 0.001). Both stimuli elevated plasma VWF levels in a manner representative of thrombotic or pro-inflammatory conditions such as arterial thrombosis. Even under these conditions, BT200 potently inhibited VWF activity and VWF-dependent platelet function, but higher BT200 concentrations were required for comparable effects relative to the unstimulated state.

Target-mediated exposure enhancement: a previously unexplored limit of TMDD.

Target-mediated drug disposition (TMDD) is often observed for targeted therapeutics, and manifests as decreases in clearance and volume of distribution with increasing dose as a result of saturable, high affinity target binding. In the present work, we demonstrate that classically defined TMDD is just one of the characteristic features of the system. In fact, for molecules with rapid non-specific elimination relative to target-mediated elimination, binding to target may actually lead to improved exposure at sub-saturating doses. This feature, which we refer to as target-mediated exposure enhancement (TMEE), produces the opposite trend to classical TMDD, i.e., with increasing dose levels, clearance and volume of distribution will also increase. The general model of TMDD was able to well-characterize the pharmacokinetics of two molecules that display TMEE, ALX-0081 and linagliptin. Additional fittings using the commonly reported TMDD model approximations revealed that both the quasi-equilibrium and quasi-steady-state approximations were able to well-describe TMEE; however, the Michaelis-Menten approximation was unable to describe this behavior. With the development of next-generation therapeutics with high affinity for target and rapid non-specific elimination, such as antibody fragments and peptides, this previously unexplored limit of TMDD is anticipated to become increasingly relevant for describing pharmacokinetics of investigational therapeutics.

Severe primary refractory thrombotic thrombocytopenic purpura (TTP) in the post plasma exchange (PEX) and rituximab era.

Acute acquired thrombotic thrombocytopenic purpura (TTP) requires prompt recognition and initiation of plasma exchange (PEX) therapy and immunosuppression. When PEX fails, mortality nears 100%, making finding an effective treatment crucial. Primary refractory TTP occurs when initial therapies fail or if exacerbations occur during PEX therapy, both signifying the need for treatment intensification to achieve clinical remission. Rituximab helps treat most of the refractory TTP cases, except those that are severely refractory. A paucity of studies guiding severely refractory TTP makes management arbitrary and individualised, highlighting the value of isolated reports. We present an extremely rare case of primary refractory TTP with an insufficient platelet response to numerous types of treatments, including emerging therapies such as caplacizumab, on the background of repeated PEX and immunosuppressive therapies.

Beyond plasma exchange: novel therapies for thrombotic thrombocytopenic purpura.

The advent of plasma exchange has dramatically changed the prognosis of acute thrombotic thrombocytopenic purpura (TTP). Recent insights into TTP pathogenesis have led to the development of novel therapies targeting pathogenic anti-ADAMTS13 antibody production, von Willebrand factor (VWF)-platelet interactions, and ADAMTS13 replacement. Retrospective and prospective studies have established the efficacy of rituximab as an adjunct to plasma exchange for patients with acute TTP, either upfront or for refractory disease. Relapse prevention is a major concern for survivors of acute TTP, and emerging data support the prophylactic use of rituximab in patients with persistent or recurrent ADAMTS13 deficiency in clinical remission. Capalcizumab, a nanobody directed against domain A1 of VWF that prevents the formation of VWF-platelet aggregates, recently completed phase 2 (TITAN) and 3 (HERCULES) trials with encouraging results. Compared with placebo, caplacizumab shortened the time to platelet recovery and may protect against microthrombotic tissue injury in the acute phase of TTP, though it does not modify the underlying immune response. Other promising therapies including plasma cell inhibitors (bortezomib), recombinant ADAMTS13, N-acetyl cysteine, and inhibitors of the VWF-glycoprotein Ib/IX interaction (anfibatide) are in development, and several of these agents are in prospective clinical studies to evaluate their efficacy and role in TTP. In the coming years, we are optimistic that novel therapies and international collaborative efforts will usher in even more effective, evidence-based approaches to address refractory acute TTP and relapse prevention.

Platelet receptors as therapeutic targets: Past, present and future.

Anti-platelet drugs reduce arterial thrombosis after plaque rupture and erosion, prevent stent thrombosis and are used to prevent and treat myocardial infarction and ischaemic stroke. Some of them may also be helpful in treating less frequent diseases such as thrombotic thrombocytopenic purpura. The present concise review aims to cover current and future developments of anti-platelet drugs interfering with the interaction of von Willebrand factor (VWF) with glycoprotein (GP) Ibα, and directed against GPVI, GPIIb/IIIa (integrin αIIbß3), the thrombin receptor PAR-1, and the ADP receptor P2Y12. The high expectations of having novel antiplatelet drugs which selectively inhibit arterial thrombosis without interfering with normal haemostasis could possibly be met in the near future.

The chimeric monoclonal antibody MHCSZ-123 against human von Willebrand factor A3 domain inhibits high-shear arterial thrombosis in a Rhesus monkey model.

BACKGROUND: SZ-123, a murine monoclonal antibody that targets the human von Willebrand factor (VWF) A3 domain and blocks the binding of collagen, is a powerful antithrombotic. In a Rhesus monkey model of thrombosis, SZ-123 had no side effects, such as bleeding or thrombocytopenia. METHODS: The mouse/human chimeric version of SZ-123, MHCSZ-123, was developed and maintained inhibitory capacities in vitro and ex vivo after injection into monkeys. CHO-S cells were selected for stable expression of MHCSZ-123. Cell clones with high levels of MHCSZ-123 expression were screened with G418 then adapted to serum-free suspension culture. The antithrombotic effect of MHCSZ-123 on acute platelet-mediated thrombosis was studied in monkeys where thrombus formation was induced by injury and stenosis of the femoral artery, which allowed for cyclic flow reductions (CFRs). CFRs were measured in the femoral artery of anesthetized Rhesus monkeys before and after intravenous administration of MHCSZ-123. Ex vivo VWF binding to collagen, platelet aggregation, platelet counts, and template bleeding time were used as measurements of antithrombotic activity. In addition, plasma VWF and VWF occupancy were measured by ELISA. RESULTS: Injection of 0.1, 0.3, and 0.6 mg/kg MHCSZ-123 significantly reduced CFRs by 29.4%, 57.9%, and 73.1%, respectively. When 0.3 and 0.6 mg/kg MHCSZ-123 were administered, 46.6%-65.8% inhibition of ristocetin-induced platelet aggregation was observed between 15 and 30 min after injection. We observed minimal effects on bleeding time, minimal blood loss, and no spontaneous bleeding or thrombocytopenia. CONCLUSIONS: The VWF-A3 inhibitor MHCSZ-123 significantly reduced thrombosis in Rhesus monkeys and appeared to be safe and well tolerated.

Treatment of thrombotic thrombocytopenic purpura beyond therapeutic plasma exchange.

Daily therapeutic plasma exchange (TPE) transformed the historically fatal prognosis of acquired, anti-ADAMTS13 antibody-mediated thrombotic thrombocytopenic purpura (TTP), leading to the current overall survival rates of 80%-85%. However, relapses occur in ~40% of patients and refractory disease with fatal outcomes still occurs. In this context, the introduction of rituximab has probably been the second major breakthrough in TTP management. Rituximab is now routinely recommended during the acute phase, typically in patients with a suboptimal response to treatment, or even as frontline therapy, with high response rates. In more severe patients, salvage strategies may include twice-daily TPE, pulses of cyclophosphamide, vincristine, as well as splenectomy in more desperate cases. In this life-threatening disease, relapse prevention represents a major goal. Persistent severe acquired ADAMTS13 deficiency in patients who are otherwise in remission is associated with a high risk of relapse and preemptive treatment with rituximab may be considered in this context. In the coming years, the TTP therapeutic landscape should be enriched by original strategies stemming from clinical experience and new agents that are currently being evaluated in large, ideally international, clinical trials. Promising agents under evaluation include N-acetylcysteine, bortezomib, recombinant ADAMTS13, and inhibitors of the glycoprotein-Ib/IX-von Willebrand factor axis.

Inherited and acquired thrombotic thrombocytopenic purpura (TTP) in adults.

Thrombotic thrombocytopenic purpura (TTP) is a clearly defined entity of thrombotic microangiopathies (TMAs), a heterogeneous group of disorders characterized by microangiopathic hemolytic anemia with red cell fragmentation, thrombocytopenia, and organ dysfunction due to disturbed microcirculation. TTP is characterized by a severe deficiency of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), an enzyme responsible for physiological cleavage of von Willebrand factor (VWF). Organ dysfunction can be severe and life threatening, and immediate start of appropriate therapy is necessary to avoid permanent damage or death. The therapeutic options, however, are often limited to symptomatic measures, and are not standardized or based on high scientific evidence. During the last years, not only considerable progress has been made in better diagnosis of TTP, but also new therapeutic strategies have been established. Initial treatment still is based on plasma exchange and symptomatic measures to protect organ function, but new concepts (immunosuppression, targeted anti-VWF or anticomplement therapy, and replacement with recombinant enzymes) are currently under development.

Acquired von Willebrand syndrome with a type 2B phenotype: diagnostic and therapeutic dilemmas.

In this report, we provide evidence of an acquired von Willebrand syndrome (AVWS) with a type 2B phenotype rather than the expected type 1 or 2A. The patient was referred prior to surgical removal of a fibrous mass within the maxillary sinus. His first bleeding 7 years earlier following a retinal tear had been complicated by monocular blindness. Several mucocutanous bleedings followed. Hematological investigations revealed von Willebrand factor (VWF):Ag 91 IU/ml, factor VIII 86 IU/ml, VWF:RCo 34 IU/ml and profound thrombocytopenia with platelet clumping. VWF multimer analysis showed a loss of high-molecular-weight multimers and his plasma aggregated normal platelets under low ristocetin concentration, consistent with type 2B von Willebrand disease (VWD). Sequencing of VWF exon 28 and of the platelet GP1BA gene to investigate the possibility of platelet-type VWD failed to reveal mutations. Serum protein electrophoresis showed a monoclonal IgG protein and led to the diagnosis of monoclonal gammopathy of unknown significance (MGUS), raising suspicion of an AVWS. Over 2 years, he experienced severe gingival bleedings and traumatic intracerebral hemorrhage. Following debridement of the sinus mass, the patient required 20 units of packed red blood cells, despite high-dose Humate-P, continuous Amicar and twice-daily platelet transfusions. Bleeding finally ceased following infusion of activated factor VIIa. A history of prior uncomplicated vasectomy and tendon laceration, no family history of bleeding, the inability to identify a causative mutation in either exon 28 VWF or platelet GP1BA and the MGUS led to diagnosis of AVWS with a type 2B phenotype. This case highlights the difficulties in assigning a diagnosis and the management of bleeding in a patient with an atypical presentation of AVWS.

No inhibitor development after continuous infusion of factor concentrates in subjects with bleeding disorders undergoing surgery: a prospective study.

Inhibitor development against von Willebrand factor, factor VIII or factor IX is one of the most severe complications of treating patients with von Willebrand's disease (VWD), haemophilia A or haemophilia B respectively. Continuous infusion of factor concentrate has been implicated as a risk factor for inhibitor development. This prospective study investigated inhibitor development after continuous infusion of factor concentrate for surgical procedures in subjects with VWD or a severe form of haemophilia (factor activity <1%). Observations were made on the occurrence of inhibitor formation, adverse events and virus seroconversions. Main inclusion criteria comprised a negative history of inhibitors to replacement factor concentrate, ≥ 50 exposure days to factor concentrate and anticipated surgery requiring replacement factor coverage for ≥ 3 days. Therapy began with a bolus dose of 30-50 IU kg(-1) body weight of factor concentrate followed by continuous infusion with 3-4 IU kg(-1) h(-1) . Continuous infusion dose of factor concentrate was adjusted based on factor levels measured at least once daily. In 46 subjects included in the study to date, no inhibitors have been identified at discharge or follow-up (3-4 weeks after surgery), and no thrombotic events or postoperative wound infections occurred. All subjects underwent surgery without major blood loss, and hemostatic efficacy was generally rated 'excellent'. The results of the current study are promising, although the number of subjects is too small to make a definitive statement about the incidence of inhibitor development during continuous infusion of factor concentrate. Therefore, this study will be continued.

Mechanisms of xenogeneic baboon platelet aggregation and phagocytosis by porcine liver sinusoidal endothelial cells.

BACKGROUND: Baboons receiving xenogeneic livers from wild type and transgenic pigs survive less than 10 days. One of the major issues is the early development of profound thrombocytopenia that results in fatal hemorrhage. Histological examination of xenotransplanted livers has shown baboon platelet activation, phagocytosis and sequestration within the sinusoids. In order to study the mechanisms of platelet consumption in liver xenotransplantation, we have developed an in vitro system to examine the interaction between pig endothelial cells with baboon platelets and to thereby identify molecular mechanisms and therapies. METHODS: Fresh pig hepatocytes, liver sinusoidal and aortic endothelial cells were isolated by collagenase digestion of livers and processing of aortae from GTKO and Gal+ MGH-miniature swine. These primary cell cultures were then tested for the differential ability to induce baboon or pig platelet aggregation. Phagocytosis was evaluated by direct observation of CFSE labeled-platelets, which are incubated with endothelial cells under confocal light microscopy. Aurintricarboxylic acid (GpIb antagonist blocking interactions with von Willebrand factor/vWF), eptifibatide (Gp IIb/IIIa antagonist), and anti-Mac-1 Ab (anti-α(M)ß(2) integrin Ab) were tested for the ability to inhibit phagocytosis. RESULTS: None of the pig cells induced aggregation or phagocytosis of porcine platelets. However, pig hepatocytes, liver sinusoidal and aortic endothelial cells (GTKO and Gal+) all induced moderate aggregation of baboon platelets. Importantly, pig liver sinusoidal endothelial cells efficiently phagocytosed baboon platelets, while pig aortic endothelial cells and hepatocytes had minimal effects on platelet numbers. Anti-MAC-1 Ab, aurintricarboxylic acid or eptifibatide, significantly decreased baboon platelet phagocytosis by pig liver endothelial cells (P<0.01). CONCLUSIONS: Although pig hepatocytes and aortic endothelial cells directly caused aggregation of baboon platelets, only pig liver endothelial cells efficiently phagocytosed baboon platelets. Blocking vWF and integrin adhesion pathways prevented both aggregation and phagocytosis.

Engraftment of human adipose derived stem cells delivered in a hyaluronic acid preparation in mice.

PURPOSE: To evaluate the implant of human adipose derived stem cells (ADSC) delivered in hyaluronic acid gel (HA), injected in the subcutaneous of athymic mice. METHODS: Control implants -HA plus culture media was injected in the subcutaneous of the left sub scapular area of 12 athymic mice. ADSC implants: HA plus ADSC suspended in culture media was injected in the subcutaneous, at the contra lateral area, of the same animals. With eight weeks, animals were sacrificed and the recovered implants were processed for extraction of genomic DNA, and histological study by hematoxilin-eosin staining and immunufluorescence using anti human vimentin and anti von Willebrand factor antibodies. CONTROLS: Not visualized at the injection site. An amorphous substance was observed in hematoxilin-eosin stained sections. Human vimentin and anti von Willebrand factor were not detected. No human DNA was detected. ADSC implants - A plug was visible at the site of injection. Fusiform cells were observed in sections stained by hematoxilin- eosin and both human vimentin and anti von Willebrand factor were detected by immunofluorescence. The presence of human DNA was confirmed. CONCLUSION: The delivery of human adipose derived stem cells in preparations of hyaluronic acid assured cells engraftment at the site of injection.

Engraftment of human adipose derived stem cells delivered in a hyaluronic acid preparation in mice / Implante de células tronco do tecido adiposo humano numa preparação de ácido hialurônico em camundongos

PURPOSE: To evaluate the implant of human adipose derived stem cells (ADSC) delivered in hyaluronic acid gel (HA), injected in the subcutaneous of athymic mice. METHODS: Control implants -HA plus culture media was injected in the subcutaneous of the left sub scapular area of 12 athymic mice. ADSC implants: HA plus ADSC suspended in culture media was injected in the subcutaneous, at the contra lateral area, of the same animals. With eight weeks, animals were sacrificed and the recovered implants were processed for extraction of genomic DNA, and histological study by hematoxilin-eosin staining and immunufluorescence using anti human vimentin and anti von Willebrand factor antibodies. RESULTS: Controls: Not visualized at the injection site. An amorphous substance was observed in hematoxilin-eosin stained sections. Human vimentin and anti von Willebrand factor were not detected. No human DNA was detected. ADSC implants - A plug was visible at the site of injection. Fusiform cells were observed in sections stained by hematoxilin- eosin and both human vimentin and anti von Willebrand factor were detected by immunofluorescence. The presence of human DNA was confirmed. CONCLUSION: The delivery of human adipose derived stem cells in preparations of hyaluronic acid assured cells engraftment at the site of injection.

OBJETIVO: Avaliar o implante de células tronco do tecido adiposo humano (CTTAH) em gel de ácido hialurônico (AH), injetados no tecido subcutâneo de camundongos atímicos. MÉTODOS: Implantes controle - HA com meio de cultura foram injetados no tecido subcutâneo da região infraescapular esquerda de 12 camundongos atímicos. Implantes de CTTAH: HA com CTTAH suspensas em meio de cultura foi injetado no subcutâneo da região contra lateral, dos mesmos animais. Com oito semanas, os animais foram sacrificados e os implantes recuperados foram processados para extração de DNA genômico, estudo histológico por coloração por hematoxilina eosina e imnuoflurescência utilizando anticorpos anti vimentina humana e anti fator de von Willebrand. RESULTADOS: Controles - implantes não visualizados no local da injeção. Uma substância amorfa foi observada nos cortes corados por hematoxilina eosina. Vimentina humana e fator anti von Willebrand não foram identificados. DNA humano não foi detectado. Implantes de CTTAH - Uma massa era visível no local da injeção. Células fusiformes foram observadas nos corte corados com hematoxilina eosina. Tanto vimentina humana quanto fator de von Willebrand foram identificados pela imunofluorescência. A presença de DNA humano foi confirmada. CONCLUSÃO: O implante de células tronco do tecido adiposo humano em veículo de ácido hialurônico gel assegurou a manutenção das células no local do implante.

ARC15105 is a potent antagonist of von Willebrand factor mediated platelet activation and adhesion.

OBJECTIVE: We investigated the stability, pharmacokinetic, and pharmacodynamic profile of the 2(nd) generation anti-von Willeband factor aptamer ARC15105. METHODS AND RESULTS: Platelet plug formation was measured by collagen/adenosine diphosphate-induced closure time with the platelet function analyzer-100 and platelet aggregation by multiple electrode aggregometry. Platelet adhesion was measured on denuded porcine aortas and in a flow chamber. Aptamer stability was assessed by incubation in nuclease rich human, monkey, and rat serum for up to 72 hours. Pharmacokinetic and pharmacodynamic profiles were tested in cynomolgus monkeys after IV and SC administration. The median IC(100) and IC(50) to prolong collagen/adenosine diphosphate-induced closure timewere 27 nmol/L and 12 nmol/L, respectively. ARC15105 (1.3 µmol/L) completely inhibited ristocetin-induced platelet aggregation in whole blood (P<0.001), but also diminished collagen, ADP, arachidonic acid, and thrombin receptor activating peptide-induced platelet aggregation to some extent (P<0.05). ARC15105 (40 nmol/L) decreased platelet adhesion by >90% on denuded porcine aortas (P<0.001), which was comparable to the degree of inhibition obtained with abciximab. ARC15105 (100 nmol/L) also inhibited platelet adhesion to collagen under arterial shear in a flow chamber by >90% (P<0.001). The IV and SC terminal half-lives in cynomolgus monkeys were 67 and 65 hours, respectively, and the SC bioavailability was ≈98%. Allometric scaling estimates the human T(1/2) would be ≈217 hours. Pharmacodynamic analysis confirms that ARC15105 inhibits von Willeband factor activity >90% in blood samples taken 300 hours after a 20 mg/kg IV or SC dose in monkeys. CONCLUSIONS: The potency, pharmacokinetic profile, and SC bioavailability of ARC15105 support its clinical investigation for chronic inhibition of von Willeband factor -mediated platelet activation.

Calmodulin antagonists induce platelet apoptosis.

Calmodulin (CaM) antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. In platelets, CaM has been found to bind directly to the cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet functions. However, it is still unknown whether CaM antagonists induce platelet apoptosis. Here we show that CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), tamoxifen (TMX), and trifluoperazine (TFP) induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, and phosphatidylserine exposure. CaM antagonists did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP-, botrocetin-, and alpha-thrombin-induced platelet aggregation, platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonists. Furthermore, cytosolic Ca(2+) levels were obviously elevated by both W7 and TMX, and membrane-permeable Ca(2+) chelator BAPTA-AM significantly reduced apoptotic events in platelets induced by W7. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis. The elevation of the cytosolic Ca(2+) levels may be involved in the regulation of CaM antagonists-induced platelet apoptosis.

The aptamer ARC1779 blocks von Willebrand factor-dependent platelet function in patients with thrombotic thrombocytopenic purpura ex vivo.

BACKGROUND: In thrombotic thrombocytopenic purpura (TTP), ultralarge von Willebrand factor (VWF) multimers bind platelet (PLT) glycoprotein Ib and lead to the formation of disseminated fibrin-poor, VWF-rich PLT thrombi. The aptamer ARC1779 blocks binding of the VWF A1 domain to PLT glycoprotein Ib. We evaluated whether ARC1779 inhibits the excessive VWF activity and VWF-mediated PLT function in patients with TTP. STUDY DESIGN AND METHODS: We studied the ex vivo concentration response curves for ARC1779 on PLT function analyzer (PFA-100, Dade Behring) and cone-and-plate analyzer (CPA, Impact-R) PLT function tests, agonist-induced PLT aggregation, and VWF activity of TTP patients (n = 11, three in acute phase and eight in remission) and healthy controls (n = 44). RESULTS: VWF activity and VWF-dependent PLT plug formation were increased in TTP patients relative to healthy controls, but agonist-induced PLT aggregation was not. ARC1779 blocked collagen/adenosine 5'-diphosphate (ADP)-induced PLT plug formation as measured by PFA-100 with an inhibitory concentration (IC)(100) of approximately 1 microg/mL in citrate-anticoagulated samples and approximately 3 to 4 microg/mL in hirudin-anticoagulated samples. A similar concentration of ARC1779 was necessary to block shear-dependent PLT adhesion in both TTP patients and healthy controls using the CPA assay (IC(100) of approx. 1 microg/mL for both). ARC1779 blocked VWF activity with an IC(90) of approximately 3 to 4 microg/mL in all subjects, but did not inhibit PLT aggregation by ADP, collagen, or arachidonic acid even at concentrations much greater than those that fully inhibited VWF-dependent PLT function. CONCLUSIONS: ARC1779 potently and specifically inhibits VWF activity and VWF-dependent PLT function. ARC1779 may be a promising novel therapeutic for the treatment of TTP.