Comprehensive functional characterization of a novel ANO6 variant in a new patient with Scott syndrome.

BACKGROUND: Scott syndrome is a mild platelet-type bleeding disorder, first described in 1979, with only 3 unrelated families identified through defective phosphatidylserine (PS) exposure and confirmed by sequencing. The syndrome is distinguished by impaired surface exposure of procoagulant PS on platelets after stimulation. To date, platelet function and thrombin generation in this condition have not been extensively characterized. OBJECTIVES: Genetic and functional studies were undertaken in a consanguineous family with a history of excessive bleeding of unknown cause. METHODS: A targeted gene panel of known bleeding and platelet genes was used to identify possible genetic variants. Platelet phenotyping, flow adhesion, flow cytometry, whole blood and platelet-rich plasma thrombin generation, and specialized extracellular vesicle measurements were performed. RESULTS: We detected a novel homozygous frameshift variant, c.1943del (p.Arg648Hisfs∗23), in ANO6 encoding Anoctamin 6, in a patient with a bleeding history but interestingly with normal ANO6 expression. Phenotyping of the patient's platelets confirmed the absence of PS expression and procoagulant activity but also revealed other defects including reduced platelet δ granules, reduced ristocetin-mediated aggregation and secretion, and reduced P-selectin expression after stimulation. PS was absent on spread platelets, and thrombi formed over collagen at 1500/s. Reduced thrombin generation was observed in platelet-rich plasma and confirmed in whole blood using a new thrombin generation assay. CONCLUSION: We present a comprehensive report of a patient with Scott syndrome with a novel frameshift variant in AN06, which is associated with no platelet PS exposure and markedly reduced thrombin generation in whole blood, explaining the significant bleeding phenotype observed.

Endogenous thrombin potential and time-dependent thrombin generation parameters are independent risk factors for mortality in the general population.

BACKGROUND: Thrombin generation (TG) is used as a global test of coagulation and is an indicator of thrombosis and bleeding risk. Until now, data on the association of TG and mortality are inconclusive. OBJECTIVES: We investigated the association between TG and mortality in the prospective Moli-sani cohort (n = 21 920). METHODS: TG was measured using calibrated automated thrombinography using PPP-Reagent Low. Lag time (LT), endogenous thrombin potential (ETP), peak height, time-to-peak (TTP), and velocity index were quantified. The association of TG and mortality was studied by Cox regression and adjusted for sex, age, body mass index, smoking, contraceptives, and medical history (cardiovascular diseases, hypertension, hypercholesterolemia, diabetes, and cancer). RESULTS: LT and TTP were 4.1 ± 1.0 minutes and 6.6 ± 1.5 minutes, on average. The peak height was 364 ± 88 nM, velocity index was 163 ± 63 nM/min, and ETP was 1721 ± 411 nM·min. ETP was negatively associated with all-cause mortality (hazard ratio [HR], 0.86; 95% CI, 0.81-0.92; P < .001). Subjects in the lowest quintile of the ETP (ETPQ1) had a 1.3-fold higher mortality rate. Additionally, a high TTP/LT ratio was negatively associated with mortality (HR, 0.71; 95% CI, 0.57-0.89; P = .003). Individuals in quintile 1 of the TTP/LT ratio had a 1.4-fold higher mortality rate compared with the remainder of the cohort. Subjects that were both in ETPQ1 and TTP/LTQ1 had a 1.8-fold higher mortality rate, regardless of whether they reported history of cardiovascular disease at baseline (HR, 1.61 [CI: 1.07-2.42]) or not (HR, 1.89 [CI: 1.51-2.36]). CONCLUSION: Low ETP and TTP/LT ratios are independent risk factors for all-cause mortality in the general population.

Altered whole blood thrombin generation and hyperresponsive platelets in patients with pancreatic cancer.

BACKGROUND: Thromboembolic disease is a major complication in patients with pancreatic ductal adenocarcinoma (PDAC). Patients with PDAC often have altered blood cell counts, which are associated with venous thromboembolism (VTE) development. The high thrombotic risk in patients with PDAC may be partially caused by procoagulant blood cells. OBJECTIVES: The aim of this study was to compare blood cell-dependent coagulation between patients with PDAC (n = 18) and healthy controls matched for age and sex (n = 18). METHODS: Thrombin generation (TG) was measured in whole blood (WB) and plasma. The capacity of platelets to release granules (PGRCs) was measured in WB. We explored the occurrence of thromboembolic events in patients with PDAC during a 6-month follow-up. RESULTS: Patients showed an increased endogenous thrombin potential in WB compared with controls. This difference was not observed in plasma, indicating a procoagulant effect of blood cells. Both in WB and plasma, the lag time was prolonged in patients compared with controls. Patients had hyperresponsive platelets, with a shorter time to peak granule release. Of the 18 patients with PDAC, 4 developed a venous thromboembolism (22%) and 1 developed an arterial thrombosis (6%). A shorter lag time in WB, but not in plasma, and an increased PGRC were associated with thromboembolic events. CONCLUSION: Patients with PDAC have an increased and delayed WB TG coagulation profile compared with controls. A shorter lag time in WB TG and increased PGRC are associated with the incidence of thromboembolic events. Platelets appear to be key players in thrombosis development. Measuring hemostasis in WB could improve thrombosis risk estimation in patients with PDAC.

Low factor XIII levels and altered fibrinolysis in patients with multiple myeloma.

BACKGROUND: Acquired factor FXIII (FXIII) deficiency can be immune- or non-immune mediated and may cause severe bleeding symptoms. The incidence of acquired FXIII deficiency and its etiology in patients with multiple myeloma (MM) are poorly understood. OBJECTIVES: To assess FXIII levels and the balance of fibrinolysis in newly diagnosed, untreated MM and monoclonal gammopathy of undetermined significance (MGUS) patients. METHODS: FXIII activity, mixing studies, FXIII-A2B2 antigen, total FXIII-B antigen were measured in platelet-poor plasma from 17 untreated MM patients, 33 untreated MGUS patients, and 30 age and sex-matched healthy controls. Besides routine laboratory measurements, the balance of coagulation and fibrinolysis was evaluated using quantitative fibrin monomer (FM) test, thrombin-antithrombin assay, α2-antiplasmin activity, plasmin-α2-antiplasmin (PAP) complex, D-dimer, plasmin generation assay, clot lysis assay, and ClotPro-TPA test. RESULTS: FXIII-A2B2 levels were significantly lower in MM patients compared to controls [median (IQR):14.6 (11.2-19.4) vs. 21.8 (17.1-26.4) mg/L, p = 0.0015], whereas total FXIII-B did not differ between groups. Decrease in FXIII activity was parallel to the decrease in FXIII-A2B2. An immune-mediated inhibitory mechanism was ruled out. Free/total FXIII-B was significantly higher in MM patients compared to MGUS and healthy controls, suggesting an etiology of FXIII-A consumption. In MM and MGUS patients, FM, D-dimer, and PAP complex were significantly elevated compared to controls, indicating hypercoagulability and ongoing fibrinolysis. CONCLUSIONS: Low FXIII levels due to consumption were observed in MM patients at diagnosis. Hypercoagulability and ongoing fibrinolysis were detected in MM and MGUS, indicating that a disturbed hemostasis balance is already present in the latter benign condition.

Crucial roles of red blood cells and platelets in whole blood thrombin generation.

Red blood cells (RBCs) and platelets contribute to the coagulation capacity in bleeding and thrombotic disorders. The thrombin generation (TG) process is considered to reflect the interactions between plasma coagulation and the various blood cells. Using a new high-throughput method capturing the complete TG curve, we were able to compare TG in whole blood and autologous platelet-rich and platelet-poor plasma to redefine the blood cell contributions to the clotting process. We report a faster and initially higher generation of thrombin and shorter coagulation time in whole blood than in platelet-rich plasma upon low concentrations of coagulant triggers, including tissue factor, Russell viper venom factor X, factor Xa, factor XIa, and thrombin. The TG was accelerated with increased hematocrit and delayed after prior treatment of RBC with phosphatidylserine-blocking annexin A5. RBC treatment with ionomycin increased phosphatidylserine exposure, confirmed by flow cytometry, and increased the TG process. In reconstituted blood samples, the prior selective blockage of phosphatidylserine on RBC with annexin A5 enhanced glycoprotein VI-induced platelet procoagulant activity. For patients with anemia or erythrocytosis, cluster analysis revealed high or low whole-blood TG profiles in specific cases of anemia. The TG profiles lowered upon annexin A5 addition in the presence of RBCs and thus were determined by the extent of phosphatidylserine exposure of blood cells. Profiles for patients with polycythemia vera undergoing treatment were similar to that of control subjects. We concluded that RBC and platelets, in a phosphatidylserine-dependent way, contribute to the TG process. Determination of the whole-blood hypo- or hyper-coagulant activity may help to characterize a bleeding or thrombosis risk.

Hyperresponsive Platelets and a Reduced Platelet Granule Release Capacity Are Associated with Severity and Mortality in COVID-19 Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is often associated with mild thrombocytopenia and increased platelet reactivity. OBJECTIVE: The aim of the current study was to investigate the adenosine triphosphate (ATP) release kinetics of platelets in hospitalized SARS-CoV-2-infected patients. METHODS: We studied time-dependent platelet activation in whole blood by monitoring the ATP release kinetics upon stimulation with a PAR1 receptor agonist in 41 hospitalized critically ill COVID-19 patients, 47 hospitalized noncritically ill COVID-19 patients, and 30 healthy controls. RESULTS: Our study demonstrated that platelets of critically ill COVID-19 patients were hyper-responsive with a shorter platelet response time (PRT) and a reduced platelet granule release capacity (GRC), probably due to chronic activation. The median PRT of COVID-19 patients admitted to the critical care unit was 10 and 7 seconds shorter than the median PRT in healthy controls and noncritical COVID-19 patients, respectively. Both PRT and GRC were also associated with D-dimer (Spearman r [r s] = -0.51, p < 0.0001 and r s = -0.23, p < 0.05), C-reactive protein (CRP) (r s = -0.59, p < 0.0001 and r s = -0.41, p < 0.01), and neutrophil-to-lymphocyte ratio (NLR) (r s = -0.42, p < 0.0001 and r s = -0.26, p < 0.05). Moreover, an increased PRT and a reduced GRC were associated with an increased mortality (odds ratio [OR]: 18.8, 95% confidence interval [CI]: 6.5-62.8, p < 0.0001 and OR: 4.0; 95% CI: 1.6-10.4, p < 0.01). These relationships remained significant after adjustment for age, sex, D-dimer, CRP, and NLR. CONCLUSION: Using an accessible agonist-induced platelet granule ATP release assay, we show that platelet hyper-responsiveness and reduced platelet GRC in COVID-19 patients were associated with critical illness and mortality.

Population-wide persistent hemostatic changes after vaccination with ChAdOx1-S.

Various vaccines were developed to reduce the spread of the Severe Acute Respiratory Syndrome Cov-2 (SARS-CoV-2) virus. Quickly after the start of vaccination, reports emerged that anti-SARS-CoV-2 vaccines, including ChAdOx1-S, could be associated with an increased risk of thrombosis. We investigated the hemostatic changes after ChAdOx1-S vaccination in 631 health care workers. Blood samples were collected 32 days on average after the second ChAdOx1-S vaccination, to evaluate hemostatic markers such as D-dimer, fibrinogen, α2-macroglobulin, FVIII and thrombin generation. Endothelial function was assessed by measuring Von Willebrand Factor (VWF) and active VWF. IL-6 and IL-10 were measured to study the activation of the immune system. Additionally, SARS-CoV-2 anti-nucleoside and anti-spike protein antibody titers were determined. Prothrombin and fibrinogen levels were significantly reduced after vaccination (-7.5% and -16.9%, p < 0.0001). Significantly more vaccinated subjects were outside the normal range compared to controls for prothrombin (42.1% vs. 26.4%, p = 0.026) and antithrombin (23.9% vs. 3.6%, p = 0.0010). Thrombin generation indicated a more procoagulant profile, characterized by a significantly shortened lag time (-11.3%, p < 0.0001) and time-to-peak (-13.0% and p < 0.0001) and an increased peak height (32.6%, p = 0.0015) in vaccinated subjects compared to unvaccinated controls. Increased VWF (+39.5%, p < 0.0001) and active VWF levels (+24.1 %, p < 0.0001) pointed toward endothelial activation, and IL-10 levels were significantly increased (9.29 pg/mL vs. 2.43 pg/mL, p = 0.032). The persistent increase of IL-10 indicates that the immune system remains active after ChAdOx1-S vaccination. This could trigger a pathophysiological mechanism causing an increased thrombin generation profile and vascular endothelial activation, which could subsequently result in and increased risk of thrombotic events.

Increased BMI and Blood Lipids Are Associated With a Hypercoagulable State in the Moli-sani Cohort.

The coagulation system can be assessed by the thrombin generation (TG) assay, and increased TG peak height, endogenous thrombin potential (ETP), and velocity index are associated with an increased risk of thrombosis. Obesity had been reported to increase TG and is associated with dyslipidemia, which also predisposes to atherosclerotic cardiovascular disease (CVD). However, the effect of the blood lipid profile on TG has not been studied extensively. To gain more insight into the associations of TG, body mass index (BMI) and lipid profile, we studied TG in relation to these parameters in a large Italian population cohort, the Moli-sani study (N = 22,546; age ≥ 35 years; 48% men). TG was measured in plasma samples collected at the enrollment of subjects in the Moli-sani study. TG was triggered with 1 or 5 pM tissue factor, and TG parameters lag time, peak, ETP, time-to-peak (TTP) and velocity index (VI). Additionally, thrombomodulin was added to assess the function of the activated protein C system during TG. In both women and men, overweight (BMI 25-30 kg/m2) and obesity (BMI > 30 kg/m2) were significantly associated with higher ETP, peak and VI (all p < 0.001). High total cholesterol, triglycerides and LDL-cholesterol levels were significantly associated with increased ETP and peak (all p < 0.001). Linear regression analysis revealed that the ETP is positively associated with both plasma LDL and HDL cholesterol levels, whereas the velocity index is positively associated with HDL cholesterol. Additionally, ETP, peak and VI were significantly associated with the plasma triglycerides content. In conclusion, our study shows significant associations of high BMI and blood lipid levels with increased TG parameters, and this hypercoagulability may partly explain the increased risk of CVD in individuals with obesity and/or dyslipidemia.

Patients With Multiple Myeloma Have a Disbalanced Whole Blood Thrombin Generation Profile.

Background: Multiple myeloma (MM) is associated with a high prevalence of bleeding and an increased risk of thrombo-embolism. MM patients have reduced platelet- and red blood cell (RBC) numbers in blood, which may indicate that the paradoxical hemostasis profile is a consequence of a disturbed platelet and RBC homeostasis. Objectives: To get better insight in the disbalanced hemostasis of MM patients. Methods: We conducted a case-control study on the whole blood (WB) coagulation profiles of 21 MM patients and 21 controls. We measured thrombin generation (TG) in WB and platelet poor plasma (PPP) of MM patients and controls. Results: In WB-TG, we observed that the median time to the thrombin Peak was 52% longer in MM patients than in controls, while the median endogenous thrombin potential until the Peak (ETPp) was 39% higher in MM-patients than in controls. In line with these findings, the levels of platelets, RBCs, white blood cells and agonist induced platelet activation were decreased in MM patients compared to controls. The plasma TG experiments showed no differences between MM-patients and controls. Conclusion: Patients with MM have a disturbed blood cell metabolism and a disbalanced WB-TG profile. This disbalance may explain the paradoxically high prevalence of bleeding symptoms in MM patients vs. an increased thrombosis risk. There was no disturbance observed in plasma TG, indicating that blood cells are the major determinants for the disbalanced hemostasis in MM patients.

Platelet Activation via Glycoprotein VI Initiates Thrombin Generation: A Potential Role for Platelet-Derived Factor IX?

Collagen triggers coagulation via activation of factor (F) XII. In a platelet-rich environment, collagen can also trigger coagulation independently of FXII. We studied a novel mechanism of coagulation initiation via collagen-dependent platelet activation using thrombin generation (TG) in platelet-rich plasma. Collagen-induced coagulation is minimally affected by active-site inactivated FVIIa, anti-FVII antibodies, or FXIIa inhibition (corn trypsin inhibitor). Activation of platelets via specific glycoprotein (GP) VI agonists initiates TG, FX activation, and fibrin formation. To determine the platelet-derived trigger of coagulation, we systematically reconstituted factor-deficient plasmas with washed platelets. TG triggered by GPVI-activated platelets was significantly affected in FIX- and FVIII-deficient plasma but not in FVII- and FXII-deficient plasma. In a purified system composed of FX and FVIII, we observed that absence of FIX was compensated by GPVI-activated platelets, which could be inhibited by an anti-FIX antibody, suggesting FIXa activity from activated platelets. Furthermore, with the addition of FVIII in FIX-deficient plasma, TG induced by GPVI-activated platelets was restored, and was inhibited by the anti-FIX antibody. In conclusion, GPVI-activated platelets initiate TG, probably via platelet-derived FIXa activity.

Differences in thrombin and plasmin generation potential between East African and Western European adults: The role of genetic and non-genetic factors.

BACKGROUND: Geographic variability in coagulation across populations and their determinants are poorly understood. OBJECTIVE: To compare thrombin (TG) and plasmin (PG) generation parameters between healthy Tanzanian and Dutch individuals, and to study associations with inflammation and different genetic, host and environmental factors. METHODS: TG and PG parameters were measured in 313 Tanzanians of African descent living in Tanzania and 392 Dutch of European descent living in the Netherlands and related to results of a dietary questionnaire, circulating inflammatory markers, genotyping, and plasma metabolomics. RESULTS: Tanzanians exhibited an enhanced TG and PG capacity, compared to Dutch participants. A higher proportion of Tanzanians had a TG value in the upper quartile with a PG value in the lower/middle quartile, suggesting a relative pro-coagulant state. Tanzanians also displayed an increased normalized thrombomodulin sensitivity ratio, suggesting reduced sensitivity to protein C. In Tanzanians, PG parameters (lag time and TTP) were associated with seasonality and food-derived plasma metabolites. The Tanzanians had higher concentrations of pro-inflammatory cytokines, which correlated strongly with TG and PG parameters. There was limited overlap in genetic variation associated with TG and PG parameters between the two cohorts. Pathway analysis of genetic variants in the Tanzanian cohort revealed multiple immune pathways that were enriched with TG and PG traits, confirming the importance of co-regulation between coagulation and inflammation. CONCLUSIONS: Tanzanians have an enhanced TG and PG potential compared to Dutch individuals, which may relate to differences in inflammation, genetics and diet. These observations highlight the importance of better understanding of the geographic variability in coagulation across populations.

Osteoprotegerin modulates platelet adhesion to von Willebrand factor during release from endothelial cells.

BACKGROUND: Platelet-binding Von Willebrand Factor (VWF) strings assemble upon stimulated secretion from endothelial cells. OBJECTIVES: To investigate the efficiency of platelet binding to multi-molecular VWF bundles secreted from endothelial cells and to investigate the role of osteoprotegerin, a protein located in Weibel-Palade bodies that interacts with the VWF platelet binding domain. METHODS: The nanobody VWF/AU-a11 that specifically binds to VWF in its active platelet-binding conformation was used to investigate the conformation of VWF. RESULTS: Upon stimulated secretion from endothelial cells, VWF strings were only partially covered with platelets, while a VWD-type 2B mutation or ristocetin enhanced platelet binding by 2-3-fold. Osteoprotegrin, reduces platelet adhesion to VWF by 40% ± 18% in perfusion assays. siRNA-mediated down-regulation of endothelial osteoprotegerin expression resulted in a 1.8-fold increase in platelet adhesion to VWF strings. Upon viral infection, there is a concordant rise in VWF and osteoprotegerin plasma levels. Unexpectedly, no such increase was observed in plasma of desmopressin-treated hemophilia A-patients. In a mouse model, osteoprotegerin expression was low in liver endothelial cells of vehicle-treated mice, and concanavalin A-treatment increased VWF and osteoprotegerin expression 4- and 40-fold, respectively. This increase was translated in a 30-fold increased osteoprotegerin/VWF ratio in plasma. CONCLUSIONS: Release of VWF from endothelial cells opens the platelet-binding site, irrespective of the presence of flow. However, not all available platelet-binding sites are being occupied, suggesting some extent of regulation. Part of this regulation involves endothelial proteins that are co-secreted with VWF, like osteoprotegerin. This regulatory mechanism may be of more relevance under inflammatory conditions.

Aprotinin Inhibits Thrombin Generation by Inhibition of the Intrinsic Pathway, but is not a Direct Thrombin Inhibitor.

Background Aprotinin is a broad-acting serine protease inhibitor that has been clinically used to prevent blood loss during major surgical procedures including cardiac surgery and liver transplantation. The prohemostatic properties of aprotinin likely are related to its antifibrinolytic effects, but other mechanisms including preservation of platelet function have been proposed. Aim Here we assessed effects of aprotinin on various hemostatic pathways in vitro, and compared effects to tranexamic acid(TXA), which is an antifibrinolytic but not a serine protease inhibitor. Methods We used plasma-based clot lysis assays, clotting assays in whole blood, plasma, and using purified proteins, and platelet activation assays to which aprotinin or TXA were added in pharmacological concentrations. Results Aprotinin and TXA dose-dependently inhibited fibrinolysis in plasma. Aprotinin inhibited clot formation and thrombin generation initiated via the intrinsic pathway, but had no effect on reactions initiated by tissue factor. However, in the presence of thrombomodulin, aprotinin enhanced thrombin generation in reactions started by tissue factor. TXA had no effect on coagulation. Aprotinin did not inhibit thrombin, only weakly inhibited the TF-VIIa complex and had no effect on platelet activation and aggregation by various agonists including thrombin. Aprotinin and TXA inhibited plasmin-induced platelet activation. Conclusion Pharmacologically relevant concentrations of aprotinin inhibit coagulation initiated via the intrinsic pathway. The antifibrinolytic activity of aprotinin likely explains the prohemostatic effects of aprotinin during surgical procedures. The anticoagulant properties may be beneficial during surgical procedures in which pathological activation of the intrinsic pathway, for example by extracorporeal circuits, occurs.

Assessing Plasmin Generation in Health and Disease.

Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The kinetics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood until the recent development of assays to quantify these metrics. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of PG by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases. This review provides an overview of available PG assays to directly measure the kinetics of plasmin formation and inhibition in human and mouse plasmas and focuses on their applications in defining the role of plasmin in diseases, including angioedema, hemophilia, rare bleeding disorders, COVID-19, or diet-induced obesity. Moreover, this review introduces the PG assay as a promising clinical and research method to monitor antifibrinolytic medications and screen for genetic or acquired fibrinolytic disorders.

Vascular activation is a strong predictor of mortality in coronavirus disease 2019 patients on the ICU.

Respiratory failure in coronavirus disease 2019 (COVID-19) patients is one of the most frequent causes for referral to the ICU. A significant percentage of these patients does not survive the infection due to thromboembolic complications. Furthermore, the vascular system seems also to be involved in the pathogenesis. To investigate the role of hemostasis and endothelium on the outcome of COVID-19 patients admitted to the ICU. Blood was drawn from 16 ICU COVID-19 patients for hemostatic analysis. Patients were followed-up till discharge (n = 11) or death (n = 5). Parameters related to both coagulation and fibrinolysis, though disturbed, were not associated with mortality. Contrarily, activated Von Willebrand factor was increased and ADAMTS13 levels were decreased by two-fold in nonsurvivors compared with survivors. Our data established the involvement of the Von Willebrand factor-ADAMTS13 axis in the COVID-19 pathogenesis, thereby demonstrating that these plasma proteins seem to be strong predictors for ICU mortality.

Interplay between platelets and coagulation.

Haemostasis stops bleeding at the site of vascular injury and maintains the integrity of blood vessels through clot formation. This regulated physiological process consists of complex interactions between endothelial cells, platelets, von Willebrand factor and coagulation factors. Haemostasis is initiated by a damaged vessel wall, followed with a rapid adhesion, activation and aggregation of platelets to the exposed subendothelial extracellular matrix. At the same time, coagulation factors aggregate on the procoagulant surface of activated platelets to consolidate the platelet plug by forming a mesh of cross-linked fibrin. Platelets and coagulation mutually influence each other and there are strong indications that, thanks to the interplay between platelets and coagulation, haemostasis is far more effective than the two processes separately. Clinically this is relevant because impaired interaction between platelets and coagulation may result in bleeding complications, while excessive platelet-coagulation interaction induces a high thrombotic risk. In this review, platelets, coagulation factors and the complex interaction between them will be discussed in detail.

Flow cytometric analysis of platelet function to improve the recognition of thrombocytopathy.

INTRODUCTION: Light transmission aggregometry (LTA) is the gold standard for diagnosing bleeding disorders. Although LTA is laborious, requires large volumes of blood and is relatively insensitive to small changes in platelet function, there is still no competing alternative approach to replace LTA for the diagnosis of platelet bleeding disorders. MATERIALS AND METHODS: This study investigates the correlation between flow cytometry-based whole blood platelet activation test (WB-PACT) and LTA and whether WB-PACT is of additional value for the identification of bleeding disorders. In total, 161 patients with suspected bleeding diathesis were tested. RESULTS: A correlation of 0.41 between LTA and WB-PACT was found, and there was agreement between tests in 62% of cases (κ = 0.23). The WB-PACT is of additional value to LTA to detect platelet function disorders (PFD) as 10 patients with elevated bleeding score (BS) were detected with WB-PACT, 4 with LTA and 7 patients were positive with both tests. Interestingly, in contrast to LTA, WB-PACT has an additional option to detect VWF disfunctions. CONCLUSION: WB-PACT may have added value for the routine diagnostic work-up in patients who need to have platelet function tested.

Acute exacerbations of COPD are associated with a prothrombotic state through platelet-monocyte complexes, endothelial activation and increased thrombin generation.

INTRODUCTION: Patients with chronic obstructive pulmonary disease (COPD) are at increased risk for cardiovascular events, particularly following an acute exacerbation (AE-COPD). Exacerbations are associated with increased systemic inflammation, which may drive coagulation. This prospective cohort study aimed to determine how an AE-COPD affects platelet activation, the endothelium, plasmatic coagulation and fibrinolysis, and its association with systemic inflammation. MATERIALS AND METHODS: Fifty-two patients with an AE-COPD were included. Blood samples at admission, at day 3 of treatment and at convalescence were available for 32 patients. Platelet-monocyte complex (PMC) formation, monocyte Mac-1 expression and platelet (re)activity (P-selectin expression, αIIbß3 activation) were measured by flow cytometry. Von Willebrand Factor (VWF), thrombin generation (TG) and clot lysis time (CLT) were determined as measures of endothelial activation, plasmatic coagulation and fibrinolysis, respectively. RESULTS: Exacerbations were associated with increased PMCs (MFI 31.3 vs 23.8, p = 0.004) and Mac-1 (MFI 38.2 vs 34.8, p = 0.006) compared to convalescence, but not with changes in platelet (re)activity. VWF (antigen, activity, active fraction) and TG (peak, ETP and velocity index) were all significantly higher during AE-COPD compared to convalescence. PMCs, Mac-1, VWF and TG were positively associated with systemic inflammation (CRP). CLT was prolonged in AE-COPD patients with systemic inflammation. Moreover, platelet hyperreactivity on admission was associated with an increased risk for exacerbation relapse. CONCLUSIONS: Acute exacerbations are associated with an inflammation-associated prothrombotic state, characterized by increased PMCs, endothelial activation and plasmatic coagulation. Our findings provide direction for future studies on biomarkers predicting the risk of exacerbation relapse and cardiovascular events.