Elisabeth C. M. van Pampus

Decreased responsiveness and development of activation markers of PLTs stored in plasma

BACKGROUND:  Circulating PLTs have a low activation state and high responsiveness, which ensures adequate hemostatic activity at sites of vessel wall damage. PLTs collected for transfusion purposes preferably have retained these properties to restore impaired hemostasis with thrombocytopenia.

The incidence of recurrent venous thromboembolism in carriers of factor V Leiden is related to concomitant thrombophilic disorders

Summary. The duration of anticoagulant treatment after a first episode of venous thromboembolism primarily depends on the risk of recurrence. Variability of recurrence rates in factor (F) V Leiden carriers may be due to concomitant thrombophilic disorders. A retrospective study was performed in 329 FV Leiden carriers with a history of venous thromboembolism (262 probands, 67 relatives). The annual rate of first recurrence was estimated in relatives. The contribution of concomitant thrombophilic disorders to the recurrence rate was evaluated in probands and relatives by a nested case–control analysis in 105 matched pairs of carriers either with or without recurrence. The overall annual recurrence rate was 2·3 per 100 patient–years. The adjusted risk of recurrence for concomitant thrombophilic disorders was: 9·1 (1·3–62·8) for the FII mutation; 1·0 (0·2–4·9) for homozygosity for FV Leiden; 1·5 (0·2–9·5) for inherited deficiencies of protein C or S; 1·8 (0·7–4·9) for FVIII coagulant activity (FVIII:C) levels > 122%; 5·4 (1·6–18·6) for fasting homocysteine levels > 15·2 µmol/l; and 4·4 (1·0–18·7) for loading homocysteine levels > 45·8 µmol/l. Of these disorders, only the FII mutation and hyperhomocysteinaemia significantly increased the risk of recurrence in FV Leiden carriers. The estimated recurrence rate ranged from 0·45 per 100 patient–years after a secondary first event in the absence of concomitant disorders to 4·8 per 100 patient–years when a spontaneous first event was combined with concomitant disorders. Our study provides supportive evidence that the incidence of recurrent venous thromboembolism in heterozygous FV Leiden carriers depends on the concomitance of other thrombophilic disorders, in addition to whether the first thrombotic event occurred spontaneously.

Increased thrombin generation and fibrinogen level after therapeutic plasma transfusion: relation to bleeding.

In a clinical setting, fresh frozen plasma (FFP) is transfused to diluted patients with complicated surgery or trauma, as guided by prolonged conventional coagulation times or low fibrinogen levels. However, the limited sensitivity of these coagulation tests may restrict their use in measuring the effect of transfusion and hence predicting the risk of perioperative bleeding. We used the more sensitive, calibrated automated thrombogram (CAT) method to evaluate the result of therapeutic FFP transfusion to 51 patients with dilutional coagulopathy. Thrombin generation was measured in pre- and post-transfusion plasma samples in the presence of either platelets or phospholipids. For all patients, the transfusion led to higher plasma coagulation factor levels, a shortened activated partial thromboplastin time, and a significant increase in thrombin generation (peak height and endogenous thrombin potential). Interestingly, thrombin generation parameters and fibrinogen levels were higher in post-transfusion plasmas from patients who stopped bleeding (n = 32) than for patients with ongoing bleeding (n = 19). Plasmas from 15 of the 19 patients with ongoing bleeding were markedly low in either thrombin generation or fibrinogen level. We conclude that the thrombin generation method detects improved haemostatic activity after plasma transfusion. Furthermore, the data suggest that thrombin generation and fibrinogen are independent determinants of the risk of perioperative bleeding in this patient group.

Effects of plasma dilution on tissue factor–induced thrombin generation and thromboelastography: partly compensating role of platelets

BACKGROUND: Bleeding upon major surgery or severe trauma is treated by transfusion with crystalloids, colloids, or plasma. This treatment, however, can lead to dilutional coagulopathy and impaired hemostasis. We investigated the suitability of two integrative coagulation tests to measure the hemostatic activity of diluted plasma.

Acquired resistance to activated protein C in breast cancer patients.

Summary. In 56 women with a lymph‐node‐positive breast carcinoma and 28 matched healthy control subjects, the sensitivity to activated protein C (APC‐sr) was determined with an APC resistance test that quantifies the effect of APC on thrombin generation initiated via the extrinsic coagulation pathway. Carriers of the Factor V Leiden mutation were excluded from the study. Significant resistance to APC was found in the breast cancer patients: median APC‐sr 2·02 vs 1·03 in the healthy control subjects (P < 0·001). No difference in APC‐sr was found between patients with metastases and without metastases. In patients with metastases, protein S levels were significantly elevated compared with patients without metastases and healthy control subjects: 108·0%vs 96·0% and 94·5% (P = 0·008 and P = 0·007). The APC‐sr correlated with protein S in the control subjects and in patients without metastases but not in patients with metastases. The disturbance of the haemostatic balance probed by the tissue‐factor‐based APC resistance test might contribute to the cancer‐related hypercoagulability.

Tissue factor activity in human monocytes is regulated by plasma: implications for the high and low responder phenomenon.

The ‘high and low responder phenomenon’ of monocyte tissue factor (MTF) activity has been attributed to effects on monocytes by granulocytes, platelets and lipopolysaccharide (LPS). To study the possible contribution of plasma to the high and low responder phenomenon, we measured the MTF activity in isolated cryopreserved human monocytes from two donors (monocytes A and monocytes B) after incubation in a plasma environment depleted of granulocytes, platelets and LPS. In buffer only, MTF activity was 643 and 679 fM (fM = final concentration of tissue factor), in normal pooled plasma, it was 1478 and 1615 fM (P = 0·001), respectively, in monocytes A and in monocytes B. Incubation with individual plasma samples from healthy controls (n = 43) gave a median MTF of 1355 fM (range 1044–1976 fM) and 1329 fM (range 858–1951 fM) respectively. A plasma consistently induced a higher or lower level of MTF activity in both monocytes: r = 0·82 (P < 0·00001). Coumarin use did not influence the high and low responder phenomenon. In the absence of granulocytes, platelets and LPS, plasma determines the high and low responder phenomenon. This phenomenon is not influenced by coumarin treatment.

Novel methodology for assessment of prophylactic platelet transfusion therapy by measuring increased thrombus formation and thrombin generation.

Currently, patients developing severe thrombocytopenia during chemotherapy treatment are prophylactically transfused with platelets. We developed two platelet function tests to report the improved haemostasis in the transfused patients, which were capable of detecting aberrant responsiveness of the platelets after transfusion. First, in a whole‐blood flow test, platelet adhesion and thrombus formation were determined under high‐shear flow conditions. Second, the procoagulant function of platelets was assayed in platelet‐rich plasma by measurement of thrombin generation. Experimental conditions were established, where flow‐induced adhesion and thrombin generation test parameters increased semi‐linearly with the platelet concentration, and informed on the activation properties of platelets. The transfusion effects were evaluated for 38 thrombocytopenic patients, who were transfused with platelets stored in plasma or in synthetic medium (platelet additive solution II). In most but not all patients, transfusion resulted in increased adhesion and thrombus formation, as well as in improved platelet‐dependent coagulation. Taken together, the increase in platelet count after transfusion explained 57% of the overall improvement in platelet function. In acute graft‐versus‐host disease, thrombus formation was normal, while platelet‐dependent coagulation was higher than expected. We conclude that assessment of flow‐induced adhesion and thrombin generation in acquired thrombocytopenia adequately determines the improved haemostatic activity by transfused platelets.

Factor XIII Val34Leu and the risk of venous thromboembolism in factor V Leiden carriers

A mutation in factor XIII (Val34Leu) was reported to protect against venous thromboembolism. We evaluated the effect of Val34Leu on thrombotic risk in 352 factor V Leiden carriers who were first‐degree relatives of 132 thrombotic propositi carrying factor V Leiden. The total observation period was 2594 years in 92 Val34Leu carriers and 7444 years in 260 non‐carriers. The annual incidence of a first episode of venous thromboembolism was 0·31% in Val34Leu carriers and 0·44% in non‐carriers [relative risk (RR) for venous thromboembolism: 0·7, 95% CI 0·3–1·5]. Age‐specific RR for venous thromboembolism were (for Val34Leu carriers and non‐carriers respectively): 1·0 (95% CI 0·3–3·2) in the age group of 15–30 years, 0·4 (95% CI 0·05–3·0) in the age group of 30–45 years, 0·6 (95% CI 0·1–2·9) in the group aged 45–60 years and 0·5 (95% CI 0·06–4·5) in relatives older than 60 years. In conclusion, the impact of FXIII Val34Leu on the venous thromboembolic risk is modest, suggesting that screening for this mutation in factor V Leiden carriers is not justified.