Characterization of Procoagulant COAT Platelets in Patients with Glanzmann Thrombasthenia

Patients affected by the rare Glanzmann thrombasthenia (GT) suffer from defective or low levels of the platelet-associated glycoprotein (GP) IIb/IIIa, which acts as a fibrinogen receptor, and have therefore an impaired ability to aggregate platelets. Because the procoagulant activity is a dichotomous facet of platelet activation, diverging from the aggregation endpoint, we were interested in characterizing the ability to generate procoagulant platelets in GT patients. Therefore, we investigated, by flow cytometry analysis, platelet functions in three GT patients as well as their ability to generate procoagulant collagen-and-thrombin (COAT) platelets upon combined activation with convulxin-plus-thrombin. In addition, we further characterized intracellular ion fluxes during the procoagulant response, using specific probes to monitor by flow cytometry kinetics of cytosolic calcium, sodium, and potassium ion fluxes. GT patients generated higher percentages of procoagulant COAT platelets compared to healthy donors. Moreover, they were able to mobilize higher levels of cytosolic calcium following convulxin-plus-thrombin activation, which is congruent with the greater procoagulant activity. Further investigations will dissect the role of GPIIb/IIIa outside-in signalling possibly implicated in the regulation of platelet procoagulant activity.

Accuracy of Fibrinogen/D-dimer Ratio in Predicting the Occurrence of Coronary Slow Flow Phenomenon

BACKGROUND: Fibrinogen is a risk factor for coronary heart disease (CHD), which is an acute-phase protein that is released when inflammation occurs. Fibrinogen is an essential component in the coagulation cascade and affects hemostasis, hemorheology, platelet aggregation, and endothelial function. D-dimers are inevitable products of fibrin degradation that results from thrombin activation; XIII activated factors and plasmin. AIM: This study was aimed to explore the accuracy of the fibrinogen/D-dimer ratio in predicting the slow flow phenomenon in CHD. METHODS: The study design is a diagnostic test with a cross-sectional design to assess the accuracy of the fibrinogen/d-dimer ratio in predicting coronary slow flow phenomenon (CSFP) in CHD. The study was conducted at the Department of Internal Medicine, Faculty of Medicine, Dr. Moh Hoesin Hospital, Palembang, Indonesia. The independent variables in this study were serum fibrinogen and D-dimer levels. Meanwhile, the dependent variable is sufferers of CHD with CSFP and regular flow. All data obtained from this study were analyzed using the SPSS version 20.0 for Windows program with a 95% confidence limit. RESULTS: This study showed that there was no difference in levels of fibrinogen and D-dimer in groups with CSFP and regular flow. Fibrinogen and D-dimer do not play a significant role in the CSFP process. The various etiological parts of this possible disorder still need further exploration and detail, ranging from the role of the renin-angiotensin-aldosterone system, the function of the autonomic nervous system, to the position of other endothelial factors. CONCLUSION: This study proves that fibrinogen has specific but not sensitive predictors of CSFP. The d-dimer value is also not accurate as a CSFP predictor. This study demonstrates that the fibrinogen / d-dimer ratio is not precise as a predictor of CSFP events.

Sodium–Calcium Exchanger Reverse Mode Sustains Dichotomous Ion Fluxes Required for Procoagulant COAT Platelet Formation

Procoagulant collagen-and-thrombin (COAT)-activated platelets represent a subpopulation of activated platelets, which retain a coat of prohemostatic proteins and express phosphatidylserine on their surface. Dichotomous intracellular signaling generating procoagulant platelet activity instead of traditional aggregating endpoints is still not fully elucidated. It has been demonstrated that secondary messengers such as calcium and sodium play a critical role in platelet activation. Therefore, we developed a flow cytometric analysis to investigate intracellular ion fluxes simultaneously during generation of aggregating and procoagulant platelets. Human platelets were activated by convulxin-plus-thrombin. Cytosolic calcium, sodium, and potassium ion fluxes were visualized by specific ion probes and analyzed by flow cytometry. We observed high and prolonged intracellular calcium concentration, transient sodium increase, and fast potassium efflux in COAT platelets, whereas aggregating non-COAT platelets rapidly decreased their calcium content, maintaining higher cytosolic sodium, and experiencing lower and slower potassium depletion. Considering these antithetical patterns, we investigated the role of the sodium–calcium exchanger (NCX) during convulxin-plus-thrombin activation. NCX inhibitors, CBDMB and ORM-10103, dose-dependently reduced the global calcium mobilization induced by convulxin-plus-thrombin activation and dose-dependently prevented formation of procoagulant COAT platelets. Our data demonstrate that both NCX modes are used after convulxin-plus-thrombin-induced platelet activation. Non-COAT platelets use forward-mode NCX, thus pumping calcium out and moving sodium in, while COAT platelets rely on reverse NCX function, which pumps additional calcium into the cytosol, by extruding sodium. In conclusion, we described for the first time the critical and dichotomous role of NCX function during convulxin-plus-thrombin-induced platelet activation.

Efficacy of a Semi Automated Commercial Closed System for Autologous Leukocyte- and Platelet-Rich Plasma (l-prp) Production in Dogs: A Preliminary Study

Background: To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and determine platelet-derived growth factor isoform BB (PDGF-BB) concentration in canine leukocyte- and platelet rich plasma (L-PRP) produced using a commercial semi-automated closed system. Methods: Twenty milliliters of citrated whole blood were obtained from 30 healthy un-sedated canine blood donors and processed using a semi-automated completely closed commercial system (CPUNT 20, Eltek group, Casale Monferrato, Alessandria, Italy) according to the manufacturer’s instructions. Erythrocyte, leukocyte, and platelet counts were determined in both whole blood (WB) and resultant L-PRP. The PDGF-BB concentration was evaluated after bovine thrombin activation of 10 L-PRP samples. Results: This commercial system produced on average 2.3 ± 0.7 mL of L-PRP containing a high concentration of platelets (767,633 ± 291,001 μL, p < 0.001), with a 4.4 fold increase in platelet count, lower concentration of erythrocytes (528,600 ± 222,773 μL, p < 0.001) and similar concentration of leukocytes (8422 ± 6346 μL, p = 0.9918) compared with WB. L-PRP had an average of 3442 ± 2061 pg/mL of PDGF-BB after thrombin activation. Neutrophils, lymphocytes and monocytes average percent content in L-PRP was 14.8 ± 13.2, 71.7 ± 18.5 and 10.7 ± 6.4, respectively. Conclusion: Sterile canine L-PRP prepared using this semi-automated closed system is easy to obtain, produces a significant increase in platelet count compared to WB and contains a detectable concentration of PDGF-BB after activation. Additional in vitro and in vivo studies are needed to assess inflammatory markers concentration and the therapeutic efficacy of this L-PRP in dogs.

Thrombin activation of PAR-1 contributes to microvascular stasis in mouse models of sickle cell disease

Vaso-occlusive crisis (VOC) is the primary cause of morbidity and hospitalization in sickle cell disease (SCD); however, only 4 therapies (hydroxyurea, l-glutamine, crizanlizumab, and voxeletor) are currently approved in SCD. These agents limit the duration, severity, and frequency of crises. Activation of coagulation is a hallmark of SCD. Studies in animal models of SCD have shown that coagulation contributes to the chronic inflammation and end-organ damage associated with the disease; however, it is unknown whether coagulation directly contributes to the microvascular stasis that causes VOC. Herein, we demonstrate that inhibition of tissue factor (TF) and the downstream coagulation proteases factor Xa and thrombin significantly attenuates heme-induced microvascular stasis in mouse models of VOC. Pharmacologic inhibition of the principal thrombin receptor, protease activated receptor-1 (PAR-1), as well as deficiency of PAR-1 in all nonhematopoietic cells, also reduces stasis in sickle mice. PAR-1 deficiency was associated with reduced endothelial von Willebrand factor expression, which has been shown to mediate microvascular stasis. In addition, TF inhibition reduces lung vaso-occlusion in sickle mice mediated by arteriolar neutrophil-platelet microemboli. In sum, these results suggest that prophylactic anticoagulation might attenuate the incidence of VOC.

Novel Insights and New Developments Regarding Coagulation Revealed by Studies of the Anti-Factor IXa (Activated Factor IX)/Factor X Bispecific Antibody, Emicizumab

Emicizumab is a humanized anti-FIXa/FX (factor IXa/X) bispecific monoclonal antibody that mimics FVIIIa (activated factor VIII) cofactor function. The hemostatic efficacy of emicizumab has been confirmed in clinical studies of patients with hemophilia A, irrespective of the presence of FVIII inhibitors. Emicizumab differs in some properties from FVIIIa molecule. Emicizumab requires no activation by thrombin and is not inactivated by activated protein C, but emicizumab-mediated coagulation is regulatable and maintains hemostasis. A small amount of FIXa (activated factor IX) is required to initiate emicizumab-mediated hemostasis, whereas tissue factor/FVIIa (activated factor VII)-mediated FXa (activated factor X) and thrombin activation initiates FVIIIa-mediated hemostasis. Fibrin formation, followed by fibrinolysis, appears to be similar between emicizumab- and FVIIIa-mediated hemostasis. These results suggest possible future uses of emicizumab for treating hemorrhagic diseases other than hemophilia A and reveal previously unobservable behaviors of procoagulation and anticoagulation factors in conventional hemostasis. Here, we have reviewed novel insights and new developments regarding coagulation highlighted by emicizumab.

Single platelet variability governs population sensitivity and initiates intrinsic heterotypic behaviours

Droplet microfluidics combined with flow cytometry was used for high throughput single platelet function analysis. A large-scale sensitivity continuum was shown to be a general feature of human platelets from individual donors, with hypersensitive platelets coordinating significant sensitivity gains in bulk platelet populations and shown to direct aggregation in droplet-confined minimal platelet systems. Sensitivity gains scaled with agonist potency (convulxin>TRAP-14>ADP) and reduced the collagen and thrombin activation threshold required for platelet population polarization into pro-aggregatory and pro-coagulant states. The heterotypic platelet response results from an intrinsic behavioural program. The method and findings invite future discoveries into the nature of hypersensitive platelets and how community effects produce population level behaviours in health and disease.