Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

Platelet aggregation inhibitors from Bothrops alternatus snake venom.

Snake venoms are a complex mixture of proteins and peptides that can activate/inhibit platelet aggregation. Bothrops alternatus venom include three main families: metalloproteinases (SVMPs), serinoproteinases (SVSPs) and phospholipases A2 (PLA2s), among other minor components. In this work, we used inhibitor cocktails (containing Na2-EDTA, PMSF and/or pBPB) to investigate the effect of these three families and of baltergin (a PIII SVMP) on platelet aggregation by a turbidmetric method using a microplate reader. Cocktails 1 (active SVMPs) and 2 (active PLA2s) significantly reduced aggregation induced by ristocetin and collagen and by collagen and thrombin, respectively. Cocktail 3 (active SVSPs) showed a mild activation of aggregation, indicating the content of thrombin-like enzymes (TLEs) in this venom is low. Cocktail 4 (active minor components) displayed inhibitory effect with all agonists assayed (ristocetin, ADP, collagen and thrombin) but at higher IC50 values. Baltergin exhibited inhibitory effect when the catalytic domain was active for ristocetin-stimulated platelet aggregation and showed a non-enzymatic mechanism of inhibition when collagen was used as agonist. It was not able to disaggregate platelet thrombus. We conclude that B. alternatus venom is a source of natural inhibitors of platelet aggregation due to the action of SVMPs and PLA2s. Other minor components such as C-type lectins likely contribute to the antiplatelet effect. The interest in knowing the action of venom components on platelet function lies both in the understanding of the pathophysiology of snake bite envenomation and in their biotechnological application.

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.

OBJECTIVE: In order to study the antithrombotic effect and mechanism of tetramethylpyrazine (TMA). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Platelet dysfunction persists after trauma despite balanced blood product resuscitation.

BACKGROUND: Platelet activation and aggregation are critical to the initiation of hemostasis after trauma with hemorrhage. Platelet dysfunction is a well-recognized phenomenon contributing to trauma-induced coagulopathy. The goal of this study was to evaluate the timing and severity of platelet dysfunction in massively transfused, traumatically injured patients during the first 72 hours after injury and its association with 30-day survival. METHODS: A retrospective secondary cohort study of platelet count and function was performed using samples from the Pragmatic Randomized Optimal Platelet and Plasma Ratios trial. Platelet characteristics were measured at 8 timepoints during the first 72 hours of hospitalization and compared between 30-day survivors and nonsurvivors. Platelet counts were assessed via flow cytometry. Platelet function was analyzed with the use of serial thrombelastography and impedance aggregometry with agonists arachidonic acid, adenosine diphosphate, collagen, thrombin receptor activating peptide, and ristocetin. RESULTS: In total, 680 patients were included for analysis. Platelet counts were significantly lower from baseline to 72 hours after hospital admission with further 1.3 to 2-fold reductions noted in nonsurvivors compared to survivor patients. Platelet aggregation via adenosine diphosphate, arachidonic acid, collagen, thrombin receptor activating peptide, and ristocetin was significantly lower in nonsurvivors at all time points. The nadir of platelet aggregation was 2 to 6 hours after admission with significant improvements in viscoelastic maximum clot formation and agonist-induced aggregation by 12 hours without concomitant improvement in platelet count. CONCLUSION: Platelet aggregability recovers 12 hours after injury independent of worsening thrombocytopenia. Failure of platelet function to recover portends a poor prognosis.

N-Stearoylethanolamine Inhibits Integrin-Mediated Activation, Aggregation, and Adhesion of Human Platelets.

N-stearoylethanolamine (NSE), a lipid mediator that belongs to the N-acylethanolamine (NAE) family, has anti-inflammatory, antioxidant, and membranoprotective actions. In contrast to other NAEs, NSE does not interact with cannabinoid receptors. The exact mechanism of its action remains unclear. The aim of this study is to evaluate the action of NSE on activation, aggregation, and adhesion of platelets that were chosen as a model of cellular response. Aggregation of platelets was measured to analyze the action of NSE (10-6-10-10 M) on platelet reactivity. Changes in granularity and shape of resting platelets and platelets stimulated with ADP in the presence of NSE were monitored by flow cytometry, and platelet deganulation was monitored by spectrofluorimetry. In vivo studies were performed using obese insulin-resistant rats. Binding of fibrinogen to the GPIIb/IIIa receptor was estimated using indirect ELISA and a scanning electron microscopy (SEM). It was found that NSE inhibits the activation and aggregation of human platelets. Our results suggest that NSE may decrease the activation and subsequent aggregation of platelets induced by ristocetin, epinephrine, and low doses of ADP. NSE also reduced the binding of fibrinogen to GPIIb/IIIa on activated platelets. These effects could be explained by the inhibition of platelet activation mediated by integrin receptors: the GPIb-IX-V complex for ristocetin-induced activation and GPIIb/IIIa when epinephrine and low doses of ADP were applied. The anti-platelet effect of NSE complements its anti-inflammatory effect and allows us to prioritize studies of NSE as a potent anti-thrombotic agent. SIGNIFICANCE STATEMENT: N-stearoylethanolamine (NSE) was shown to possess inhibitory action on platelet activation, adhesion, and aggregation. The mechanism of inhibition possibly involves integrin receptors. This finding complements the known anti-inflammatory effects of NSE.

Navigating Hemostasis of Bleeding Among Children With ß-Thalassemia.

Bleeding phenotype is reported in ß-thalassemia patients. However, the underlying etiology remains elusive. We aimed to assess coagulation profile and the platelet aggregation in ß-thalassemia children with bleeding diathesis. Fifty ß-thalassemia children with a positive bleeding history were recruited. Bleeding phenotype was explored through full history taking and thorough clinical examination. Complete blood count, prothrombin time, international normalized ratio, and platelets aggregometry were performed for children with negative workup. Mucosal bleeding was manifest among most of our patients (96%). Two-third of patients had decreased aggregation with ristocetin (68%), adenine di-phosphate (64%), and arachidonic acid (64%). While half of the patients (48%) had deficient response to epinephrine. Collagen, ristocetin, and arachidonic acid induced aggregation were negatively correlated to frequency of blood transfusion (P=0.021, r=-0.325; P<0.001, r=-0.465; P=0.018, r=-0.333, respectively). Aggregation to collagen and epinephrine demonstrated a negative correlation with age (P=0.04, r=-0.287; P=0.03, r=-0.315). Deferiprone was associated with a deficient response to ristocetin and collagen when compared with deferasirox or no chelation (P=0.021 and 0.006, respectively). Impaired ristocetin response was linked to hydroxyurea (P=0.035). Platelets function defect should be considered in ß-thalassemia patients with bleeding symptoms.

The von Willebrand factor A-1 domain binding aptamer BT200 elevates plasma levels of von Willebrand factor and factor VIII: a first-in-human trial.

Von Willebrand factor (VWF) and factor VIII (FVIII) circulate in a noncovalent complex in blood and promote primary hemostasis and clotting, respectively. A new VWF A1-domain binding aptamer, BT200, demonstrated good subcutaneous bioavailability and a long half-life in non-human primates. This first-in-human, randomized, placebo-controlled, doubleblind trial tested the hypothesis that BT200 is well tolerated and has favorable pharmacokinetic and pharmacodynamic effects in 112 volunteers. Participants received one of the following: a single ascending dose of BT200 (0.18-48 mg) subcutaneously, an intravenous dose, BT200 with concomitant desmopressin or multiple doses. Pharmacokinetics were characterized, and the pharmacodynamic effects were measured by VWF levels, FVIII clotting activity, ristocetin-induced aggregation, platelet function under high shear rates, and thrombin generation. The mean half-lives ranged from 7-12 days and subcutaneous bioavailability increased dose-dependently exceeding 55% for doses of 6-48 mg. By blocking free A1 domains, BT200 dose-dependently decreased ristocetin-induced aggregation, and prolonged collagen-adenosine diphosphate and shear-induced platelet plug formation times. However, BT200 also increased VWF antigen and FVIII levels 4-fold (P<0.001), without increasing VWF propeptide levels, indicating decreased VWF/FVIII clearance. This, in turn, increased thrombin generation and accelerated clotting. Desmopressin-induced VWF/FVIII release had additive effects on a background of BT200. Tolerability and safety were generally good, but exaggerated pharmacology was seen at saturating doses. This trial identified a novel mechanism of action for BT200: BT200 dose-dependently increases VWF/FVIII by prolonging half-life at doses well below those which inhibit VWF-mediated platelet function. This novel property can be exploited therapeutically to enhance hemostasis in congenital bleeding disorders.

In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

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.

Activation of von Willebrand factor via mechanical unfolding of its discontinuous autoinhibitory module.

Von Willebrand factor (VWF) activates in response to shear flow to initiate hemostasis, while aberrant activation could lead to thrombosis. Above a critical shear force, the A1 domain of VWF becomes activated and captures platelets via the GPIb-IX complex. Here we show that the shear-responsive element controlling VWF activation resides in the discontinuous autoinhibitory module (AIM) flanking A1. Application of tensile force in a single-molecule setting induces cooperative unfolding of the AIM to expose A1. The AIM-unfolding force is lowered by truncating either N- or C-terminal AIM region, type 2B VWD mutations, or binding of a ristocetin-mimicking monoclonal antibody, all of which could activate A1. Furthermore, the AIM is mechanically stabilized by the nanobody that comprises caplacizumab, the only FDA-approved anti-thrombotic drug to-date that targets VWF. Thus, the AIM is a mechano-regulator of VWF activity. Its conformational dynamics may define the extent of VWF autoinhibition and subsequent activation under force.

Impact of electric cardioversion on platelet activation.

INTRODUCTION: Atrial fibrillation (AF) comes along with high risk of stroke. This risk continues even after re-establishing sinus rhythm with cardioversion. Aim of this study is to evaluate the contribution of electric cardioversion (EC) to platelet activation and procoagulatory tendency. METHODS: Extent of platelet activation before and after electric cardioversion was quantified using flow cytometry, impedance aggregation measurements with Multiplate®, and quantification of serum levels of platelet factor 4 (PF4) and ß-thromboglobulin (ß-TG) in patients with AF (N = 10). RESULTS: No significant differences were observed in any of the measured parameters comparing the values from before and after cardioversion. Geometric means of P-selectin expression and integrin αIIbß3 activation were 0.27 (+/- 0.07) and 2.30 (+/- 2.61) before EC and 0.28 (+/- 0.17) and 1.67 (+/- 1.82) after EC. Levels of ß-TG were 110.11 ng/ml (+/- 3.78) before and 110.51 ng/ml (+/- 2.56) after EC, levels of PF4 were 35.64 ng/ml (+/- 12.94) before and 32.40 ng/ml (+/- 4.95) after EC. Platelet aggregation triggered with adenosine diphosphate (ADP), arachidonic acid, collagen, Ristocetin, or thrombin receptor activating peptide (TRAP) revealed results within the normally expected ranges without significant changes before and after EC. DISCUSSION: Electric cardioversion has no influence on platelet activation markers which is in agreement with other studies reporting electrical cardioversion to be safe.

Platelet Dysfunction in Noonan and 22q11.2 Deletion Syndromes in Childhood.

INTRODUCTION: An underlying thrombocytopathy seems to be responsible for hemorrhagic symptoms in patients diagnosed with 22q11.2 deletion syndrome (22q11DS) or Noonan syndrome (NS). In 22q11DS, it is explained by a defect in the membrane glycoprotein (GP) complex Ib-V-IX. The cause of thrombocytopathy in NS remains unclear. AIM: The objective is to study the incidence of thrombocytopathy in pediatric patients diagnosed with 22q11DS or NS assessing the utility of ISTH-BAT questionnaire and laboratory techniques. MATERIALS AND METHODS: Prospective study between March and December 2018 in children (2-18 years old) diagnosed with 22q11DS or NS. Hemorrhagic symptoms using ISTH-BAT score, total cell blood count, platelet indices, PFA-200 closure times, and platelet aggregation were evaluated in all patients and membrane GP expression in 22q11DS patients. RESULTS: Nearly 70% of NS patients (n = 22) had a platelet aggregation defect without thrombocytopenia. A defect of platelet aggregation with adenosine diphosphate (ADP) and epinephrine was the most frequent pattern. A statistically significant inverse correlation between closure times and aggregation with arachidonic acid (p = 0.049, p = 0.043) and epinephrine (p = 0.021, p = 0.035), and ADP (p = 0.117, p = 0.05) was found. Total 5 out of 29 patients diagnosed with 22q11DS had macrothrombocytopenia; more noteworthy in older patients. Twenty-six patients showed an impairment in ristocetin-induced platelet aggregation that correlated with prolonged collagen/epinephrine (p = 0.034) and collagen/ADP (p = 0.01). A significant association between ISTH-BAT score >3 and closure times (p = 0.022, p = 0.002) and aggregation defect with ristocetin (p = 0.043) was also demonstrated. CONCLUSION: Most NS and 22q11DS patients show an impairment of platelet aggregation that correlates with closure times. In 22q11DS patients, these results were also related to hemorrhagic symptoms.

The Direct Thrombin Inhibitors Dabigatran and Lepirudin Inhibit GPIbα-Mediated Platelet Aggregation.

The direct thrombin inhibitor (DTI) dabigatran is a non-vitamin K antagonist oral anticoagulant for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation. In addition to its anti-thrombotic efficacy, dabigatran has been suggested to exert some pro-thrombotic effect due to fostering the ligation of thrombin to its high affinity platelet receptor glycoprotein (GP) Ibα in patients with atrial fibrillation. On the other hand, we provided evidence that a member of another class of DTIs, lepirudin, stimulates the inhibitory cyclic guanosine monophosphate (cGMP)/soluble guanylate cyclase pathway in human platelets. Here, we investigated the effect of lepirudin and dabigatran spiked to platelets from healthy volunteers on GPIbα-mediated platelet aggregation and agglutination. Ristocetin/von Willebrand factor (vWF)-induced aggregation of platelets in the presence or absence of plasma was significantly inhibited by lepirudin, dabigatran and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK). However, ristocetin/vWF-mediated platelet agglutination and binding of vWF to platelets were not affected by the DTIs. The anti-aggregatory effect was confirmed by using the GPIbα-specific agonist echicetin beads for human and murine platelets. DTIs diminished echicetin beads-induced Syk Y352 phosphorylation (used here as readout for an early signal occurring during echicetin-induced platelet aggregation), but did not inhibit adenosine diphosphate- or thromboxane A2-induced platelet aggregation. Thrombin was not generated in response to ristocetin/vWF or echicetin beads and therefore did not explain the inhibitory effect of the DTIs. Therapeutic concentration of lepirudin and dabigatran did not affect significantly platelet vasodilator-stimulated phosphoprotein S239 phosphorylation or cGMP and cyclic adenosine monophosphate levels. These data suggest that the DTIs, lepirudin and dabigatran, impair platelet activation measured during platelet aggregation induced by ristocetin/vWF or echicetin beads.

Whole blood ristocetin-induced platelet impedance aggregometry does not reflect clinical severity in patients with type 1 von Willebrand disease.

BACKGROUND: The haemorrhagic phenotype in patients with von Willebrand disease (VWD) is heterogeneous, and assays of von Willebrand factor ristocetin cofactor activity (VWF:RCo) do not always reflect clinical severity, especially in those individuals classed as type 1 VWD. Recent studies have shown that whole blood ristocetin-induced platelet agglutination (WB-RIPA) using an easy-to-use analyzer, Multiplate® platelet impedance technique, could be informative as a diagnostic test in VWD, although inconsistencies were evident in patients with the type 1 disorder, possibly associated with clinical symptoms. AIM: To investigate the relationship between WB-RIPA, bleeding scores (BS) and VWF-related measurements in type 1 VWD. METHODS: WB-RIPA assay using the Multiplate® was performed using whole blood from 55 patients with type 1 VWD. BS was determined using a standardized questionnaire. RESULTS: WB-RIPA values were significantly lower in type 1 VWD than in healthy controls (P < 0.0001). Weak correlations were apparent between WB-RIPA and VWF:RCo or VWF antigen (VWF:Ag; r = 0.22 or 0.28, respectively). There were significant differences in VWF:RCo (P = 0.036) and VWF:Ag (P = 0.0013) between patients with BS ≥4 (defined as abnormal bleeding tendency) and BS <4 (defined as no abnormal bleeding tendency), respectively. However, no significant difference was observed in WB-RIPA between the BS ≥4 group and BS <4 group. Overall, VWD patients with a WB-RIPA level >70 U did not seem to have an abnormal bleeding tendency, but low levels of WB-RIPA did not correlate with BS. CONCLUSION: WB-RIPA did not reflect clinical severity in type 1 VWD patients.

Platelet aggregation response in immune thrombocytopenia patients treated with romiplostim.

The thrombopoietin receptor agonist romiplostim is used for the long-term treatment of chronic immune thrombocytopenia (ITP). ITP patients have an increased thrombotic risk, which could be exacerbated if romiplostim increased platelet hyperreactivity or caused spontaneous platelet aggregation. To investigate this possibility, this study examined platelet function in romiplostim-treated ITP patients and healthy subjects. Light transmission platelet aggregometry utilizing arachidonic acid, collagen, epinephrine, ristocetin, ADP, and saline (to assess spontaneous aggregation) was performed for each subject. In addition, the ADP AC50 (ADP concentration that induced half-maximal aggregation) was determined for each patient as a sensitive measurement of altered platelet reactivity. Fifteen ITP patients and 7 healthy subjects entered the study. All ITP patients had active disease and were receiving weekly romiplostim as the sole ITP-directed therapy. Platelet aggregation in response to the strong agonists arachidonic acid, collagen, and ristocetin was not significantly different between ITP patients and healthy subjects (P = 0.2442, P = 0.0548, and P = 0.0879, respectively). Platelet aggregation in response to weak agonists was significantly reduced in ITP patients compared with that in healthy subjects: median (range) aggregation to ADP, 45% (15-84%) versus 89% (70-95%) (P = 0.0010), and epinephrine, 21% (1.6-90%) versus 88% (79-94%) (P = 0.0085). The median AC50 of ADP was threefold higher in ITP patients versus that in healthy subjects (6.3 µM vs 2.1 µM) (P = 0.0049). Significant spontaneous aggregation was not observed in any patient. Platelets from romiplostim-treated ITP patients do not show evidence for spontaneous aggregation or hyperreactivity, but instead have a modestly reduced aggregation response to ADP and epinephrine.

Trichlorination of a Teicoplanin-Type Glycopeptide Antibiotic by the Halogenase StaI Evades Resistance.

Glycopeptide antibiotics (GPAs) include clinically important drugs used for the treatment of infections caused by Gram-positive pathogens. These antibiotics are specialized metabolites produced by several genera of actinomycete bacteria. While many GPAs are highly chemically modified, A47934 is a relatively unadorned GPA lacking sugar or acyl modifications, common to other members of the class, but which is chlorinated at three distinct sites. The biosynthesis of A47934 is encoded by a 68-kb gene cluster in Streptomyces toyocaensis NRRL 15009. The cluster includes all necessary genes for the synthesis of A47934, including two predicted halogenase genes, staI and staK In this study, we report that only one of the halogenase genes, staI, is necessary and essential for A47934 biosynthesis. Chlorination of the A47934 scaffold is important for antibiotic activity, as assessed by binding affinity for the target N-acyl-d-Ala-d-Ala. Surprisingly, chlorination is also vital to avoid activation of enterococcal and Streptomyces VanB-type GPA resistance through induction of resistance genes. Phenotypic assays showed stronger induction of GPA resistance by the dechlorinated compared to the chlorinated GPA. Correspondingly, the relative expression of the enterococcal vanA resistance gene was shown to be increased by the dechlorinated compared to the chlorinated compound. These results provide insight into the biosynthesis of GPAs and the biological function of GPA chlorination for this medically important class of antibiotic.

Perhaps it's not the platelet: Ristocetin uncovers the potential role of von Willebrand factor in impaired platelet aggregation following traumatic brain injury.

BACKGROUND: Injury to the blood-brain barrier exposes endothelium rich in von Willebrand factor (vWF), which may play a role in altered platelet aggregation following traumatic brain injury (TBI). Ristocetin is an antimicrobial substance that induces vWF-mediated aggregation of platelets. We examined these mechanisms in injured patients by measuring the aggregation response of platelets to stimulating agonists (including ristocetin) via whole-blood multiple-electrode platelet aggregometry. We hypothesized that patients with TBI have an altered platelet aggregation response to ristocetin stimulation compared with patients without TBI. METHODS: Blood was collected from 233 trauma patients without thrombocytopenia. Platelet aggregation was assessed using multiple-electrode platelet aggregometry (Multiplate). Platelet aggregation response to stimulating agonists collagen, thrombin receptor-activating peptide 6, adenosine diphosphate, arachidonic acid, and ristocetin was measured. Factor activity was measured. RESULTS: Of the 233 patients, 23% had TBI. There were no differences in platelet aggregation responses to any agonists between TBI and non-TBI patients except ristocetin. Platelet aggregation response to ristocetin stimulation was significantly lower in TBI patients (p = 0.03). Patients with TBI also had higher factor VIII activity (215% vs. 156%, p = 0.01). In multivariate analysis, there was a significant independent association of impaired platelet aggregation response to ristocetin stimulation with TBI (odds ratio, 3.05; p = 0.04). CONCLUSIONS: Given the importance of platelets in hemostasis, understanding the mechanisms of impaired platelet aggregation following injury is critical. The impaired platelet aggregation response to ristocetin stimulation and corresponding increase in factor VIII activity in TBI patients may be secondary to a TBI-induced effect on vWF quantity (due to injury-driven consumption of vWF) or vWF function with resultant increase in circulating factor VIII activity (due to impaired carrying capacity of vWF). Given there are multiple known therapies for vWF deficits including desmopressin, purified and recombinant vWF, and estrogens, these lines of investigation are particularly compelling in patients with TBI and hemorrhage. LEVEL OF EVIDENCE: Prognostic study, level II.

Whole blood ristocetin-activated platelet impedance aggregometry (Multiplate) for the rapid detection of Von Willebrand disease.

Von Willebrand disease (VWD) is the most common bleeding disorder, but no bedside tests specific for Von Willebrand factor are available. The objective of this study was to evaluate the diagnostic accuracy of whole blood ristocetin-induced platelet aggregometry (WB-RIPA) in VWD. WB-RIPA was performed in VWD patients (n=100) and healthy controls (n=17) using the Multiplate® platelet impedance aggregometry platform. The diagnostic properties of the test were described as sensitivity/specificity, positive and negative predictive value, and ROC area under the curve (AUC). Patients with VWD had impaired platelet aggregation by WB-RIPA. At a cut-off of 98 U, the test sensitivity and specificity of WB-RIPA for VWD was 0.95 and 0.53. A cut-off of 60 U provided a specificity of 1.00 with reduced sensitivity of 0.76. All patients with type 3 VWD and >90 % of patients with type 2 VWD were accurately distinguished from the controls. Incorrect classifications were attributable to patients with type 1 VWD, showing partly overlapping WB-RIPA results with healthy controls. Remarkably, these patients had lower bleeding scores and higher VWF activity than other type 1 VWD patients. Overall, WB-RIPA discriminated VWD patients from healthy controls accurately with a ROC AUC of 0.94. These results show that WB-RIPA is a promising diagnostic test for VWD, especially when timely results are required. Depending on the chosen test threshold, WB-RIPA could be clinically used as a rule out test, or to suggest patients in whom further testing for VWD is warranted.

Comparison of haemostatic function of PAS-C-platelets vs. plasma-platelets in reconstituted whole blood using impedance aggregometry and thromboelastography.

BACKGROUND AND OBJECTIVES: There are concerns about the haemostatic function of platelets stored in platelet additive solution (PAS). Aim of this study was to compare the haemostatic function of PAS-C-platelets to plasma-platelets in reconstituted whole blood. MATERIALS AND METHODS: In our experiment, whole blood was reconstituted with red blood cells, solvent-detergent (SD) plasma and either PAS-C-platelets or plasma-platelets (n = 7) in a physiological ratio. On storage days 2, 5, 8 and 13, the agonist-induced aggregation (multiple electrode aggregometry), clot formation (thromboelastography) and agonist-induced CD62P responsiveness (flow cytometry) were measured. RESULTS: Samples with PAS-C-platelets showed significantly lower aggregation than plasma-platelets when induced with adenosine diphosphate, -6 U (95% confidence interval: -8; -4) or thrombin receptor-activating protein, -15 U (-19; -10). Also when activated with collagen and ristocetin, the PAS-C-platelets showed less aggregation, although not statistically significant. All samples with PAS-C-platelets showed significantly lower agonist-induced CD62P responsiveness than samples with plasma-platelets. However, there was no difference regarding all TEG parameters. CONCLUSION: Our findings demonstrate that the function - aggregation and CD62P responsiveness - of PAS-C-platelets in reconstituted whole blood is inferior to that of plasma-platelets, which may have implications in the setting of massive transfusions.

Potent Thrombolytic Effect of N-Acetylcysteine on Arterial Thrombi.

BACKGROUND: Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N-Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization. METHODS: Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection. RESULTS: We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis. CONCLUSIONS: We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.