Ristocetin-induced Platelet Aggregation 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.

Current Diagnosis of von Willebrand Disease in Italy: 3 Years Following the Release of the International Guidelines.

The American Society of Hematology-International Society on Thrombosis and Haemostasis-National Hemophilia Foundation-World Federation of Hemophilia 2021 International Guidelines (IGL) on von Willebrand disease (VWD) have pointed out many challenges, mainly in the diagnostic approach of VWD patients. To determine the impact of these IGL on the current clinical and laboratory diagnosis of Italian VWD patients, we have recently conducted a survey among 43 centers affiliated with the Italian Association of Hemophilia Centers (AICE). Directors and colleagues responsible for the management of VWD patients were invited to report in a detailed questionnaire how IGL recommendations about the assessment of the specific activities of von Willebrand Factor (VWF) could be applied at their local sites. Results from such a survey showed that bleeding assessment tools, VWF antigen, and factor VIII procoagulant are currently in use in all centers. The automated assays for platelet-dependent VWF activity with or without ristocetin described in IGL have been used since 2021 in 37/43 (86%) centers. Among other laboratory tests, VWF collagen binding, ristocetin-induced platelet agglutination, multimeric analysis, VWF propeptide, VWF:FVIII binding assay were available in 49, 63, 26, 7, and 28% of AICE, respectively. Analyses of VWF gene defects are available only at 3/43 (7%) centers. Desmopressin (DDAVP) infusion trials at diagnosis, with measurements of VWF activities at 1 and 4 hours post-DDAVP, is currently performed at 38/43 (88%) centers. Based on this information, a simplified clinical diagnosis using a few automated tests before and after DDAVP has been proposed. Such a diagnostic approach will be validated prospectively in a large cohort of Italian VWD patients.

Reduction of ristocetin-induced platelet aggregation (RIPA) during storage despite plasma renewal: evidence for hemostatic importance of GPIbα shedding

ABSTRACT Background Platelet storage is complicated by deleterious changes, among which reduction of ristocetin-induced platelet aggregation (RIPA) has a poorly understood mechanism. The study elucidates the mechanistic roles of all the possible players in this process. Research design and methods PRP-platelet concentrates were subjected to RIPA, collagen-induced platelet aggregation (CIPA), and flowcytometric analysis of GPIbα and PAC-1 binding from days 0 to 5 of storage. Platelet-poor plasma was subjected to colorimetric assays for glucose/LDH evaluation and automatic analyzer to examine VWF antigen and activity. Results From day three of platelet storage, reducing CIPA but not RIPA was correlated with the reduction of both metabolic state and integrin activity. RIPA reduction was directly related to the decreased levels of total-content/expression of GPIbα, and inversely related to its shedding levels during storage. Re-suspension of 5-day stored platelet in fresh plasma compensated CIPA, but not RIPA. VWF concentration and its activity did not change during storage while they had no correlation with RIPA. Conclusions This study identified the irreversible loss of platelet GPIbα, but not VWF status, as the primary cause of the storage-dependent decrease of RIPA. Unlike CIPA, this observation was not compensated by plasma refreshment, suggesting that some evidence of PSL may not be recovered after transfusion.

Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module

AbstractActivation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

A Compound Heterozygosis of Two Novel Mutations in vWF Exacerbates vWD in a Chinese Pedigree.

von Willebrand disease (vWD), caused by mutations in the von Willebrand factor (vWF) coding gene, is a disease characterized by abnormal coagulation activity and a severe tendency for hemorrhage. Therefore, identifying mutations in vWF is important for diagnosing congenital vWD. We studied a 23-year-old male vWD patient and his parents. Clotting methods were used to determine activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen (FIB) levels, FVIII activity. Chromogenic substrate method was used to determine vWF antigen and activity. The platelet count was determined. Mutations were searched using whole-exome sequencing and certified by Sanger sequencing. Clinical data, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen levels, FX activity, FX antigen levels, and the platelet count were collected. A mixing study was performed to eliminate the presence of coagulation factor inhibitors and lupus anticoagulants. Mutations were screened by using whole-exome sequencing (WES) and were verified by using Sanger sequencing. The proband showed severely decreased vWF antigen, vWF activity, and FVIII activity. RIPA (RISTO-CETIN-induced platelet aggregation) was 0%. Data from WES showed that the proband carried compound heterozygous variants vWF: NM_000552.5 (c.3213C>A p.Cys1071Ter) and vWF: NM_000552.5 (c.6598+2T>C). The proband's mother carried variant vWF: NM_000552.5 (c.3213C>A p.Cys1071Ter) while the proband's father carried variant vWF: NM_000552.5 (c.6598+2T>C). All laboratory test indexes of the proband's parents, including vWF antigen, vWF activity, and FVIII activity, were within the normal ranges. We identified a compound heterozygosis with two novel mutations in vWF (c.3213C>A, c.6598+2T >C) in a family pedigree, and our results demonstrate that the compound heterozygous mutations probably exacerbate vWD.

Evaluation of a diagnostic platelet aggregation test strategy for platelet rich plasma samples with low platelet counts.

Light transmission aggregometry (LTA) is important for diagnosing platelet function disorders (PFD) and von Willebrand disease (VWD) affecting ristocetin-induced platelet aggregation (RIPA). Nonetheless, data is lacking on the utility of LTA for investigating thrombocytopenic patients and platelet rich plasma samples with low platelet counts (L-PRP). Previously, we developed a strategy for diagnostic LTA assessment of L-PRP that included: (1) acceptance of referrals/samples, regardless of thrombocytopenia severity, (2) tailored agonist selection, based on which are informative for L-PRP with mildly or severely low platelet counts, and (3) interpretation of maximal aggregation (MA) using regression-derived 95% confidence intervals, determined for diluted control L-PRP (C-L-PRP). To further evaluate the L-PRP LTA strategy, we evaluated findings for a subsequent patient cohort. Between 2008 and 2021, the L-PRP strategy was applied to 211 samples (11.7% of all LTA samples) from 192 unique patients, whose platelet counts (median [range] × 109 /L) for blood and L-PRP were: 105 [13-282; 89% with thrombocytopenia] and 164 [17-249], respectively. Patient-L-PRP had more abnormal MA findings than simultaneously tested C-L-PRP (p-values <0.001). Among patients with accessible electronic medical records (n = 181), L-PRP LTA uncovered significant aggregation abnormalities in 45 (24.9%), including 18/30 (60%) with <80 × 109 platelets/L L-PRP, and ruled out PFD, and VWD affecting RIPA, in others. The L-PRP LTA strategy helped diagnose VWD affecting RIPA, Bernard Soulier syndrome, familial platelet disorder with myeloid malignancy, suspected ITGA2B/ITGB3-related thrombocytopenia, and acquired PFD. Diagnostic LTA with L-PRP, using a strategy that considers thrombocytopenia severity, is feasible and informative.

Pathogen Reduced Cryoprecipitated Fibrinogen Complex (IFC) and Cryoprecipitated AHF Contain Von Willebrand Factor with Comparable Binding to Collagen and Support Shear-Induced Platelet Thrombus Formation

Traumatic hemorrhage is a leading cause of morbidity and mortality and is a potentially preventable cause of death. Trauma-induced coagulopathy (TIC) exhibits complex pathophysiology. Fibrinogen (FIB) deficiency, a major mediator of TIC, is an independent predictor of mortality; and one of the first factors to reach critically low levels during hemorrhage. Early supplementation of FIB using cryoprecipitated AHF (Cryo) administration in resuscitation has been shown to improve outcomes in the treatment of TIC. Unlike FIB concentrates, Cryo prepared from conventional plasma is enriched in von Willebrand factor (VWF), factor VIII, factor XIII, and fibronectin that are impacted by TIC in addition to FIB. However, Cryo has a limited post thaw shelf-life of 4-6 hours at room temperature. This causes logistical impediments for early clinical application in trauma often resulting in use late in resuscitation with significant wastage. Pooling of individual Cryo units to achieve therapeutic doses increases donor exposure with risk of bacterial and viral transfusion transmitted infections (TTI). FDA approved Pathogen Reduced Cryoprecipitated Fibrinogen Complex (IFC) manufactured from Amotosalen/UVA treated plasma (PR plasma) is an alternative FIB source that has a 5-day post-thaw shelf-life at RT with reduced TTI risk. Due to its longer post-thaw shelf-life, it can be thawed in advance so that it is readily available and can be administered early in massive transfusion protocols with significantly lower wastage. VWF is a key component of Cryo and plays a major role in hemostasis during resuscitation with TIC. Here we have compared VWF contained in Cryo and IFC in various functional assays. At equimolar concentrations as assessed by measured VWF antigen, IFC and Cryo supported similar ristocetin-induced platelet aggregation. Accordingly, VWF multimeric composition, particularly presence of the largest molecular species, was similar in IFC and Cryo. We then evaluated the ability of VWF in IFC and Cryo compared to native blood to support platelet adhesion and platelet-rich thrombus formation onto a fibrillar type I collagen surface exposed to a linearly increasing shear rate gradient (0 t0 20,000 s -1) in the direction of flow from inlet to outlet of a Hele-Shaw flow chamber. Briefly, human blood, collected in EDTA, Lepirudin and supplemented with PGE 1 was centrifuged to separate blood cells from plasma. Red cells and platelets were washed to remove all plasma VWF and reconstituted to original hematocrit with Tyrode buffer at pH 7.4 spiked with either Cryo or IFC up to 1 U/ml of VWF. Platelets in native blood and in the different mixtures were fluorescently labeled with 3,3'-dihexyloxacarbocyanine iodide (DiOC 6) for visualization, and image sequences were acquired at different shear rates along the gradient. Images were analyzed to quantify platelet adhesion (the proportion of the field of view covered by platelets) and aggregation (i.e., the number and area of aggregates) at any given shear rate. As a negative control, blood reconstituted with buffer instead of a VWF source was used to ascertain that plasma VWF was effectively removed. As positive control, normal donor blood was used without washing out VWF (native blood). At all tested shear rates, the total surface coverage (1 pixel = 0.099 µm 2) by platelets was comparable with VWF in AHF or IFC added to washed blood and native blood (Figure). Of note, the individual size of platelet aggregates tended to increase with increasing shear rates and the number of individual aggregates tended to decrease. Specific monoclonal antibodies against VWF A1 and A3 domains - blocking interaction with GPIbα and collagen, respectively - markedly reduced the number and size of platelet aggregates over the surface. These results indicate that VWF derived from IFC, comparable to Cryo and native blood, retains functions strictly required to support platelet adhesion and aggregation at extremely elevated levels of shear rate/stress. The figure shows the size where 1 pixel = 0.099 µm 2 of each individual platelet thrombus on the surface.

Antioxidant and antithrombotic activities of the Distichochlamys citrea leaves extract

Distichochlamys citrea (DC) is an endemic ginger species used in treating associated-heart diseases in traditional medicine in Vietnam. However, scientific evidence to support the local use of this plant was limited. The present study aimed to investigate the antioxidant and antithrombotic activities of D. citrea extracts for the first time. The antioxidant activity of DC extracts was assessed by scavenging DPPH radical and measuring their total phenolic content (TPC). The antithrombotic activity was evaluated by inhibiting platelet aggregation and prolonging blood coagulation. Volatile components elucidated by GC-MS were docked with typical platelet receptors, including COX-1 and P2Y12. Results showed that the methanol extract of D. citrea exhibited a stronger DPPH scavenging ability (IC50 = 0.33 ± 0.00 mg/mL) and a higher TPC (8.09 ± 0.21%) than other extracts (p &lt; 0.05). On the other hand, the hexane extract of D. citrea (DC-HX) had a remarkable inhibiting impact on ADP-, collagen- and ristocetin–induced platelet aggregation in a dose-dependent manner (Pearson’s correlation, r &gt; 0.90, p &lt; 0.05). In contrast, this extract did not lengthen the clotting time through any factors, such as PT (prothrombin time), and TT (thrombin time), except for APTT (activated partial thromboplastin time) at 4 mg/mL of the extract. GC-MS revealed that oxygenated hydrocarbons (54.45%) dominated the volatile profile of DC-HX, followed by sesquiterpens (37.18%) and diterpenes (6.66%). In the platelet aggregation process, several compounds in DC-HX were firmly bound to COX-1 and P2Y12, which might partly explain the significant antiaggregatory activity of this fraction. In conclusion, Distichochlamys citrea may be a potential source of active phytoconstituents for treating radicals- and cardiovascular-associated diseases.

Evaluation of Global Hemostasis Assays in Von Willebrand Disease Type 3 in Comparison with Mild to Moderate Hemophilia a

Introduction: Von Willebrand disease (VWD) is the most common inherited bleeding disorder. Type 3 VWD is the most severe form of this disease with a prevalence of about 0.5 to 1 per million in Western countries but is much more common in communities with high levels of consanguinity like South India. Type 3 VWD is characterised by undetectable Von Willebrand Factor and reduced Factor VIII levels. (1,2) Global hemostasis assays clot waveform analysis (CWA) [3,4] and thrombin generation test (TGT)[5,6] reflect the entire process of thrombin generation, and thus reflect the actual bleeding severity . Rotational Thromboelastogram (ROTEM) demonstrates the dynamics of clot formation over time. Global Hemostasis Assays is extensively studied in Hemophilia A(HA) patients, however there are very few studies which analysed the same in type 3 VWD in account of rarity of disease Our aim was to study ROTEM, TGT and APTT-CWA parameters in cases of vWD 3. We also aimed to compare global hemostasis parameters in vWD3 to those in mild to moderate hemophilia A with similar levels of Factor VIII. Materials and Methods: Retrospective study for a period of 2 years which included 27 cases of vWD3 (with undetectable vWF antigen) and 34 cases of Hemophilia A- 25 moderate and 9 mild deficiency (F VIII-5-10%). Complete coagulation workup was done which included International Society of Thrombosis and Hemostasis based Bleeding Assessment Tool(BAT) Score questionnaire, Bleeding time, Platelet Function Analyser 200, Prothrombin Time(PT), APTT, Ristocetin Cofactor, Von Willebrand Antigen, Factor assays, Ristocetin induced platelet aggregation and Global Hemostasis Assays-ROTEM, TGT and CWA. BAT score was done to assess severity of bleeding. TGT was performed by Calibrated Automated Thrombogram, CWA by photo-optical coagulometer and factor levels by one stage clot-based assays on ACL Top 750. Rotem was performed in whole blood by modified extem. TGT was could be done only on 16/34 Hemophilia A cases and 14/27 vWD3 cases. Statistical Analysis: For continuous data, the descriptive statistics mean, SD, and for non-normally distributed data median, IQR were done. Based on the normality of data, the parametric t-test or non-parametric Mann Whitney test was used to find the difference between groups. All tests were two-sided at alpha () =0.05 level of significance. All the statistical analysis was performed using Statistical Package for Social sciences (SPSS) version 21.0. Results: Results are summarized in Table 1Global hemostasis tests in vWD3 A.1)ROTEM- CT (Clotting Time) was prolonged in 44% cases, 55 % cases it was within normal range and shortened in rest. CFT (Clot Formation Time) and alpha angle were within normal range in 81.1% cases, prolonged in 0.3% cases and shortened in rest. MCF (Maximum Clot Firmness) was reduced in 2 cases, and normal in remaining cases. ML(Maximum Lysis) was within range for all cases. A.2) APTT CWA- Min1 was decreased in 25.9 % cases, rest 74% had within normal range. Min2 was decreased in 96% cases. Max 2 was decreased in all cases. A.3) TGT- Lag time was prolonged in 13/14 (93%) cases. ETP was increased in 7/14 cases (50%). Peak height was decreased in all cases. TTP was prolonged in 13/14 cases (93%). Start tail was prolonged in 12/14 cases.B) Comparison between vWD3 and Mild to Moderate Hemophilia Refer to table -1 APTT was significantly prolonged (P value 0.008) in HA group than vWD3, though Factor VIII was similar in both groups. CWA were comparable to HA cases. Min 2 was statistically lower in vWD3 compared to HA group between the 2 groups (P value- 0.018) ROTEM were comparable between the 2 groups. In TGT, the median lag time (min)was significantly more prolonged in vWD3 with median of 5.50(4.95-6.37) than Hemophilia A patients as seen with APTT. ETP and Peak Thrombin were not significantly different between the 2 groups. Time to peak and start tail were statistically lower among cases of vWD 3 and haemophilia A, p value being <0.05. CONCLUSION: Global hemostasis test in von Willebrand disease showed a hypocoagulable state in Thrombin Generation Tests and APTT CWA. ROTEM didn't show any significant change despite Factor VIII levels being low Tests which measure Factor VIII assay and tests which reflect Factor VIII activity as in global hemostasis assay do not show significant difference in patients who have low Factor VIII level irrespective of the diagnosis being von Willebrand disease or Hemophilia A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Application of microfluidic chip technology to study the inhibitory effect of tetramethylpyrazine on platelet aggregation, activation, and phosphatidylserine exposure mediated by pathological high shear rate.

In order to study the antithrombotic effect and mechanism of tetramethylpyrazine (TMA). In this study, we developed a microfluidic chip model that can mimic normal arteries and stenotic arterial vessels, and studied the inhibitory effects of TMA on platelet aggregation, activation (P-selectin, GPIIb/IIIa, monocyte-platelet aggregates) and phosphatidyl serine (PS) exposure. In addition, we also investigated the effect of TMA on ADP and ristocetin-induced platelet aggregation by turbidimetry. The results showed that TMA significantly inhibited the platelet aggregation, activation and PS exposure induced by pathological high shear rate. Under static conditions, TMA can inhibit ADP and ristocetin-induced platelet aggregation. The results indicated that TMA mainly inhibited platelet aggregation, activation and PS exposure by inhibiting the binding of von Willebrand factor (vWF) to the GPIb/IX/V complex, and partially inhibited platelet aggregation through the platelet P2Y 12 -ADP receptor pathway.

High levels of von Willebrand factor with reduced specific activities in hospitalized patients with or without COVID-19.

The COVID-19 pandemic is often accompanied by severe respiratory illness and thrombotic complications. Von Willebrand Factor (VWF) levels are highly elevated in this condition. However, limited data are available on the qualitative activity of VWF in COVID-19. We measured plasma VWF levels quantitatively (VWF antigen) and qualitatively (ristocetin-induced platelet agglutination, glycoprotein IbM (GPIbM) binding, and collagen binding). Consistent with prior reports, VWF antigen levels were significantly elevated in hospitalized patients with or without COVID-19. The GPIbM and collagen binding activity-to-antigen ratios were significantly reduced, consistent with qualitative changes in VWF in COVID-19. Of note, critically ill hospitalized patients without COVID-19 had similar reductions in VWF activity-to-antigen ratios as patients with COVID-19. Our data suggest that qualitative changes in VWF in COVID-19 may not be specific to COVID-19. Future studies are warranted to determine the mechanisms responsible for qualitative changes in VWF in COVID-19 and other critical illnesses.• VWF levels were increased in COVID-19 compared to healthy controls.• VWF activity-to-antigen ratios were decreased in COVID-19 compared to healthy controls.• There were no differences in VWF activity-to-antigen ratios between hospitalized patients with or without COVID-19.• These findings are consistent with qualitative changes in VWF in systemic inflammation which are not specific to COVID-19.• Future studies are needed to define possible roles of changes in conformation or multimer length in the qualitative changes in VWF in systemic inflammation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11239-022-02679-5.

Genomic Confirmation of the P-IIIe Subclass of Snake Venom Metalloproteinases and Characterisation of Its First Member, a Disintegrin-Like/Cysteine-Rich Protein.

Disintegrin-like/cysteine-rich (DC) proteins have long been regarded just as products of proteolysis of P-III snake venom metalloproteinases (SVMPs). However, here we demonstrate that a DC protein from the venom of Vipera ammodytes (Vaa; nose-horned viper), VaaMPIII-3, is encoded per se by a P-III SVMP-like gene that has a deletion in the region of the catalytic metalloproteinase domain and in part of the non-catalytic disintegrin-like domain. In this way, we justify the proposal of the introduction of a new subclass P-IIIe of SVMP-derived DC proteins. We purified VaaMPIII-3 from the venom of Vaa in a series of chromatographic steps. A covalent chromatography step based on thiol-disulphide exchange revealed that VaaMPIII-3 contains an unpaired Cys residue. This was demonstrated to be Cys6 in about 90% and Cys19 in about 10% of the VaaMPIII-3 molecules. We further constructed a three-dimensional homology model of VaaMPIII-3. From this model, it is evident that both Cys6 and Cys19 can pair with Cys26, which suggests that the intramolecular thiol-disulphide exchange has a regulatory function. VaaMPIII-3 is an acidic 21-kDa monomeric glycoprotein that exists in at least six N-glycoforms, with isoelectric points ranging from pH 4.5 to 5.1. Consistent with the presence of an integrin-binding motif in its sequence, SECD, VaaMPIII-3 inhibited collagen-induced platelet aggregation. It also inhibited ADP- and arachidonic-acid-induced platelet aggregation, but not ristocetin-induced platelet agglutination and the blood coagulation cascade.

Laboratory assays of VWF activity and use of desmopressin trials in the diagnosis of VWD: a systematic review and meta-analysis

Abstractvon Willebrand Disease (VWD) is associated with significant morbidity because of excessive bleeding. Early diagnosis and treatment are important to prevent and treat these symptoms. We systematically reviewed the accuracy of any von Willebrand factor (VWF) activity assay in the diagnosis and classification of patients for VWD. We searched Cochrane Central, MEDLINE, and EMBASE for eligible studies. The risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS)-2 and the certainty of evidence using the GRADE framework. We pooled estimates of sensitivity and specificity. The review included 77 studies that evaluated the use of newer tests of VWF platelet binding activity (VWF:GPIbR, VWF:GPIbM) and VWF:RCo for the diagnosis of VWD (13 studies), VWF propeptide to VWF:Ag ratio, and desmopressin trial for the diagnosis of type 1C VWD (5 studies), VWF multimer analysis and VWF:CB/VWF:Ag ratio for the classification of type 2 VWD (11 studies), genetic testing and ristocetin-induced platelet aggregation to diagnose type 2B VWD (14 studies), genetic testing and FVIII:VWF binding to diagnose type 2N VWD (17 studies). Based on available diagnostic test accuracy, there appear to be comparable test accuracy results between newer tests of platelet binding activity of VWF function and VWF:RCo. The findings of these reviews support VWF multimer analysis or VWF:CB/VWF:Ag to diagnose type 2 VWD. The desmopressin trial test with 1- and 4-hour postinfusion blood work is the test of choice to confirm increased VWF clearance in patients with suspected VWD type 1C. Additionally, genetic testing is most useful in diagnosing type 2B VWD and has a role in the diagnostic algorithm of suspected type 2N VWD.

In Vitro Comparison of Recombinant and Plasma-Derived von Willebrand Factor Concentrate for Treatment of Acquired von Willebrand Syndrome in Adult Extracorporeal Membrane Oxygenation Patients

Coagulopathic bleeding is common during adult extracorporeal membrane oxygenation (ECMO), and acquired von Willebrand syndrome is a contributing factor. We compared ECMO patient blood samples that were treated in vitro with recombinant von Willebrand Factor concentrate and plasma-derived von Willebrand Factor concentrate. Our hypothesis was that recombinant von Willebrand Factor (vWF) would have greater efficacy in increasing vWF function. Secondarily, we hypothesized that recombinant vWF would have less impact on thrombin generation. Thirty ECMO patients and 10 cardiac surgical controls were enrolled in the study. ECMO patient blood samples were treated in vitro with low- and high-dose recombinant vWFs and low- and high-dose plasma-derived vWFs. Whole blood ristocetin-induced platelet aggregation (RIPA), plasma ristocetin cofactor activity (RCo), and thrombin generation were compared between ECMO patient blood samples and control blood samples and between vWF-treated ECMO patient blood samples and nontreated samples. ECMO patient blood samples had severely reduced median RIPA compared to control samples 2 ohms (1-12 [25th-75th percentile]) vs 20 ohms (11-42) (P < .001). Treatment of ECMO patient blood samples with high-dose recombinant vWF significantly increased median RIPA to 10 ohms (2-15) (P < .001), while low-dose recombinant vWF and low- and high-dose plasma-derived vWFs did not significantly increase RIPA; 6 ohms (3-14), 4 ohms (1-13), and 6 ohms (2-10), respectively (P = .25, >.99, and >.99). Treatment with high-dose recombinant vWF and low- and high-dose plasma-derived vWFs significantly increased median plasma RCo to 4.7 international units (IU)/mL (3.7-5.9), 3.3 IU/mL (2.7-4.8), and 3.9 IU/mL (3.4-5.3), respectively, compared to controls 1.8 IU/mL (1.5-2.3) (all P < .001). Treatment with low- and high-dose plasma-derived vWFs significantly increased mean endogenous thrombin potential (6270.2 ± 2038.7 and 6313.1 ± 1913.3) compared to nontreated samples (5856.7 ± 1924.6) (P = .04 and .006), whereas treatment with low- and high-dose recombinant vWFs had no significant effect on mean endogenous thrombin potential (5776.1 ± 2087.3 and 5856.2 ± 1946.4) (P > .99 for both comparisons). In vitro treatment of ECMO patient blood samples with high-dose recombinant vWF was superior to low-dose recombinant vWF and plasma-derived vWF in terms of improving RIPA. In addition, recombinant vWF treatment did not increase endogenous thrombin potential, which may reduce overall thrombotic risk if it used to treat acquired von Willebrand syndrome in ECMO patients.

Successful pregnancy and delivery management in a patient with Bernard Soulier Syndrome.

Bernard Soulier Syndrome (BSS) is an inherited bleeding disorder characterized by macrothrombocytopenia and absence of ristocetin-induced platelet aggregation. Clinical findings vary from person to person. Most of the patients are diagnosed with muco-cutaneous bleeding such as purpura, epistaxis and gingival bleeding in early childhood. Few pregnant women with BSS are described in the literature. Management of thrombocytopenia during pregnancy and delivery requires a multidisciplinary approach. The family should be warned about the potentially life-threatening bleeding during pregnancy and the delivery and the decision about mode of delivery should be individualised, involving discussion with patient and multidisciplinary team.

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.

Von Willebrand factor multimeric assay: novel diagnostics capabilities

Introduction. Distinguishing between Von Willebrand disease (vWD) types often requires multimer gel analysis. The current techniques for vWF multimer structure are manual, complicated, non-standardized and time consuming. The aim of this study was to evaluate diagnostic capabilities of new automated vWF multimer screening assay.Materials and methods. Children with vWD, acquired von Willebrand Syndrome (aVWS) and 8 healthy donors as a control group were enrolled in this study. Von Willebrand factor antigen (vWF Ag); ristocetin cofactor activity (VWF:Rco); vWF collagen binding (VWF:CB); ristocetin-induced platelet aggregation (RIPA); factor VIII clotting activity (FVIII:C) and vWF factor VIII binding activity (vWF:FVIIIb) were performed to evaluate vWD. Multimer analysis was carried out using the commercial HYDRAGEL 5 von Willebrand Multimers kit on semi-automatic gel electrophoresis instrument HYDRASYS (SEBIA).Results. The samples from control group had 9—12 bands of vWF multimers with the same distribution as control plasma. Patients with type I vWD had the proportional decrease in the intensity of the bands with preservation of the normal distribution of the band. Patients with type III vWD reveal the complete absence of the multimer bands on the gel. Multimer analysis in type IIA shows the absence of high molecular weight multimer bands. In other patients the distribution of vWF multimers was normal against the changes in functional properties of vWF (types IIM, N). Most of the children with aVWS also revealed normal distribution of vWF multimers, however, in some patients, the slight decrease in large multimeric forms was observed visually on the gel.Conclusion. Multimer analysis allows to visualize the multimer distribution in various types of von Willebrand disease. The method is easy to perform and can be useful for distinguishing between the subtypes of vWD. But only the full test panel including genetic tests would allow the differentiantion of vWD types with high precision.

Inhibition of the Shear-Induced Platelet Microparticle Formation by a mAb Against GPIb alpha Is Associated with Protein Tyrosine Dephosphorylation and the Blockade of Filamin Cleavage.

Platelet microparticle (MP) formation during shear-induced platelet aggregation (SIPA) contributes to a relevant part of thrombin generation. A monoclonal antibody (mAb) Ib-23 against platelet GPIb alpha (provided by B.S.) inhibits von Willebrand factor binding, ristocetin-induced platelet aggregation (RIPA), shear induced MP formation and thrombin generation. Platelet calpain affects several functions including MP-formation. One of several target substrates of calpain, filamin, binds to the cytoplasmic tail of GPIbalpha and mediates the assembly of the contractile elements during platelet activation. We therefore hypothesized that the inhibitory mechanism of our mAb Ib-23 on MP formation is a “calpeptin-like effect”, i.e. an inhibition of calpain affecting the protein tyrosine phosphorylation.PPACK-anticoagulated platelet-rich plasma or washed platelets (250′000/μl) were exposed to a shear rate of 5000 sec−1 for 5 min in a cone-plate viscometer. The mAb Ib-23 was added at the RIPA-inhibiting IC80–90. The samples were fluorescence-labelled with an anti-GPIIb-PE mAb to monitor SIPA and MP formation and analyzed by flow cytometry. Resting and sheared platelets were lysed in SDS-PAGE loading buffer for the analysis of filamin degradation and protein tyrosine phosphorylation.Results: Epitope mapping showed that mAb Ib-23 binds between residues 234 and 456 on GP Ib alpha. Exposure of platelets to high shear rates induced the activation of calpain as demonstrated by the degradation of filamin, a phenomenon blocked by the mAb Ib-23. Ib-23 did not inhibit the SIPA induced tyrosine phosphorylation of high molecular weight proteins like FAK, Syk and alpha-actinin, it even weakly enhanced the tyrosine phosphorylation of these proteins. Moreover, Ib-23 prevented the dephosphorylation of low molecular weight proteins (appr. 40 and 33kDa). Annexin V-binding was unaffected by Ib-23. In contrast, abciximab, a GP IIb/IIIa antagonist, partially inhibited SIPA and annexin binding, but not MP generation. Abciximab weakly inhibited the degradation of filamin, inhibited SIPA-induced tyrosine phosphorylation of FAK and Syk, but did not prevent the tyrosine dephosphorylation of the low molecular weight proteins. Control-experiments with calpeptin demonstrated a similar inhibition of filamin degradation as with mAb Ib-23. An isotype control antibody was without effects. The intact antibody Ib-23 was more effective than the Fab2 fragment. A mAb against the FcgammaRII receptor (IV.3, to exclude a bridging of the FcgammaRII and GpIbalpha via the antibody's Fc part) did not affect MP-formation.We conclude that binding of mAb Ib-23 on the outside of GP Ib alpha inhibits SIPA-induced MP formation and thrombin generation possibly via a “calpeptin-like effect” which inhibits the activation of calpain and of protein tyrosine phosphatases.

Von Willebrand disease type Vicenza: In search of a classification for the archetype of reduced von Willebrand factor survival.

Type Vicenza von Willebrand disease (VWD) features a von Willebrand factor (VWF) with a very short half‐life, and is classified as a form of type 1 VWD. To test the appropriateness of type Vicenza VWD classification, the main features of 17 patients from eight unrelated families were analysed. They had low VWF antigen levels and function (always below 20 U/dl); ristocetin‐induced platelet aggregation sometimes normal, sometimes reduced/absent (even in the same patient); normal platelet VWF levels; an increased VWF propeptide to VWF antigen ratio (8.74 ± 1.65 vs. normal 1.04 ± 0.28) and a reduced VWF half‐life. Plasma VWF multimer levels were homogeneously reduced, and unusually large VWF multimers were sometimes present. Recombinant p.R1205H VWF showed a normal synthesis, release, function, and multimer pattern, with no ultra‐large VWF multimers. The mathematical model by Galvanin et al. was used to explore the kinetic changes in VWF after DDAVP. It showed that the release, but especially the proteolysis (k proteol 1.0−3 ± 2.5−3 vs. normal 4.5−4 ± 6.4−4) and elimination (k el 1.0−2 ± 5.2−3 vs. normal 1.1−3 ± 6.8−4) of type Vicenza VWF were significantly higher than normal. The increased elimination is consistent with the short half‐life, while the increased proteolysis was unexpected. As a shorter survival of VWF is wholly responsible for the type Vicenza VWD phenotype (VWF synthesis, structure and function are normal), it might be better to classify it as a type 2 VWD (rather than type 1) to emphasise the greater interaction with clearance receptors as a new VWF functional defect.

The aptamer BT200 blocks von Willebrand factor and platelet function in blood of stroke patients

The effect of conventional anti-platelet agents is limited in secondary stroke prevention, and their effects are blunted under high shear stress in the presence of increased levels of circulating von Willebrand factor (VWF). VWF is critically involved in thrombus formation at sites of stenotic extracranial/intracranial arteries. A third generation anti-VWF aptamer (BT200) has been generated which could be useful for secondary stroke prevention. To characterize the effects of BT200 in blood of patients with large artery atherosclerosis stroke (LAA). Blood samples were obtained from 33 patients with acute stroke or transient ischemic attack to measure inhibition of VWF activity and VWF-dependent platelet function. Patients who received clopidogrel or dual antiplatelet therapy did not differ in VWF dependent platelet function tests from aspirin treated patients. Of 18 patients receiving clopidogrel with or without aspirin, only 3 had a prolonged collagen adenosine diphosphate closure time, and none of the patients had ristocetin induced aggregation in the target range. BT200 concentration-dependently reduced median VWF activity from 178 to < 3%, ristocetin induced platelet aggregation from 40U to < 10U and prolonged collagen adenosine diphosphate closure times from 93 s to > 300 s. Baseline VWF activity correlated (r = 0.86, p < 0.001) with concentrations needed to reduce VWF activity to < 20% of normal, indicating that BT200 acts in a target concentration-dependent manner. Together with a long half-life supporting once weekly administration, the safety and tolerability observed in an ongoing phase I trial, and the existence of a reversal agent, BT200 is an interesting drug candidate.