Ilia Tamarin

A NEW METHOD FOR QUANTITATIVE ANALYSIS OF WHOLE BLOOD PLATELET INTERACTION WITH EXTRACELLULAR MATRIX UNDER FLOW CONDITIONS

A new method and device in which whole blood platelet deposition and aggregation on extracellular matrix (ECM) under defined shear conditions is quantitatively evaluated was developed. A 0.25 mL aliquot of citrated whole blood is placed on ECM and a defined shear rate is applied for 2 min using a cone and plate device. This is followed by staining and measuring the number of stained objects, the percentage of ECM surface covered with stained objects and the average size of the objects using an image analyzer. When normal blood is analyzed, platelet deposition is a shear and a time dependent process, reaching maximal levels within 2 min at high shear rate (1300 s-1) of about 20% surface coverage and average aggregate size of about 40-50 microns 2. These two parameters demonstrated positive correlation with the platelet count and the hematocrit. Studies using samples from patients with von Willebrand disease (vWD) and Glanzmanns Thrombasthenia (GT) were performed and demonstrated the ability of the new method to detect these pathological conditions. Blood samples of vWD patients showed a very low adhesion and aggregation at high shear rate as reflected by very low surface coverage (5.2%) and average particle size of single platelets (21.3 microns2). GT samples at a high shear rate demonstrated surface coverage similar to normal blood samples (21.7%) but with average particle size of single platelets (21.3 microns2). The new method is an alternative method to clinically evaluate platelet function under close to physiological conditions.

Inherited factor XI deficiency confers no protection against acute myocardial infarction

Summary.  Background and purpose: Factor XI (FXI) contributes to thrombin generation thereby affecting fibrin formation and to down regulation of fibrinolysis by activation of thrombin‐activatable fibrinolysis inhibitor (TAFI). The purpose of this study was to evaluate whether patients with severe FXI deficiency are protected against acute myocardial infarction (AMI). Methods: The incidence of AMI in patients with severe FXI deficiency (FXI activity less than 15 U dL−1) whose age was 35 years or more was compared to the incidence of AMI in age and gender matched persons of the general population. Atherosclerotic risk factors were assessed in FXI deficient patients and blood was tested for prothrombotic parameters such as FV Leiden, prothrombin G20210A, lupus anticoagulant, and platelet membrane polymorphisms. The common mutations causing FXI deficiency in Jews were also examined. Results: Of 96 patients with severe FXI deficiency (55 women and 41 men) 16 had a history of AMI (6 women and 10 men). The median age at the time of AMI was 64.5 for women and 58 for men. The calculated annual rate of AMI in men was similar to the expected in the general Israeli population, whereas in women it was almost 2‐fold higher, but this difference did not reach statistical significance. One or more atherosclerotic risk factors were observed in 13 of 16 patients (81.3%) with AMI compared to 44 of 79 patients (55.7%) without AMI (P < 0.001). The frequency distributions of platelet polymorphisms and of prothrombotic polymorphisms were not different between patients with severe FXI deficiency who experienced or not an AMI. None of the patients had lupus anticoagulant. The common genotypes which cause FXI deficiency in Jews were similarly distributed in patients with and without AMI. Conclusions: Severe FXI deficiency does not confer protection against AMI.

Plasma replacement therapy during labor is not mandatory for women with severe factor XI deficiency.

Severe factor XI deficiency is an injury-related bleeding disorder. The risk of excessive post-partum hemorrhage in affected women has so far been evaluated in a relatively small number of patients and it is uncertain whether prophylactic treatment with fresh frozen plasma or factor XI concentrate is needed during or after vaginal or cesarean delivery. We retrospectively analyzed bleeding manifestations related to vaginal and/or cesarean deliveries in a cohort of 62 women with factor XI activity < 17 U/dl and evaluated whether replacement therapy is essential. Fifty-one women had 139 vaginal deliveries, six women had 13 cesarean deliveries, and five women had seven vaginal as well as five cesarean deliveries. Forty-three of the 62 women (69.4%) never experienced post-partum hemorrhage during 93 deliveries (85 vaginal, eight cesarean). Hemorrhage occurred in 19 women, which in six women accompanied each one of their 17 vaginal deliveries. Post-partum hemorrhage had no relationship with the abnormal genotype that caused factor XI deficiency nor with factor XI level. These observations suggest that the use of fresh frozen plasma or factor XI concentrate during and/or after vaginal delivery is not mandatory in women with severe factor XI deficiency and can be reserved for patients who develop excessive hemorrhage. For women requiring cesarean section it appears that the same policy can be advocated but more observations are needed.

Cone and plate(let) analyzer: Monitoring glycoprotein IIb/IIIa antagonists and von Willebrand disease replacement therapy by testing platelet deposition under flow conditions

Exposure of citrated whole blood samples to polystyrene plates under flow conditions with the cone and plate(let) analyzer (CPA) results in surface immobilization of plasma von Willebrand factor (vWF) followed by platelet deposition. Staining the plates allows measurement of the percentage of surface covered (SC) by the adhered particles and their average size (AS). Both SC and AS parameters depend on platelet count and hematocrit level and reach maximal values after 2 minutes; only AS is shear rate dependent. Under optimal assay conditions (2 minutes at 1800 sec(-1)) normal blood samples yielded an SC of 14.9% +/- 2.5% and an AS of 39.4 +/- 5.2 microm2. Severe von Willebrand disease (eight patients) yielded low SC (5.2% +/- 2.1%), which was restored to normal by testing with vWF precoated surfaces. Glanzmanns thrombasthenia (six patients) samples demonstrated no adhesion of platelet at all. Blocking of the glycoprotein (GP) IIb/IIIa receptor by the chimeric antibody abciximab, and of the GPIb by a recombinant vWF fragment, have yielded a dose-response inhibition demonstrating the crucial role of these receptors in platelet deposition on polystyrene plates in this system. Evaluation of a patient with severe von Willebrand disease receiving replacement vWF factor VIII therapy revealed a comparable response as tested by both the CPA and the Ricof methods. In vitro testing of the GPIIb/IIIa blocking by a nonpeptidic analogue tirofiban and abciximab revealed a good correlation between the CPA test and the routine aggregometry. Eleven patients treated by abciximab after coronary angioplasty were studied by the CPA during the first 24 hours, demonstrating a marked decrease in SC and AS, with some diversity in the responses. We conclude that the CPA test is suitable for evaluation of primary hemostasis and for monitoring of anti-platelet drugs.

Recombinant activated factor VII and tranexamic acid are haemostatically effective during major surgery in factor XI-deficient patients with inhibitor antibodies

One-third of patients with severe factor XI (FXI) deficiency caused by homozygosity for null alleles develop inhibitor antibodies following exposure to plasma. Haemostasis during surgery is achievable in such patients by recombinant activated factor VII (rFVIIa) at doses used in haemophilia A patients with an inhibitor to FVIII. However, thrombosis has occurred in three of 12 such patients. In this study we discerned whether low-dose rFVIIa would secure haemostasis and cause no thrombosis in patients with severe FXI deficiency and an inhibitor during surgery. In vitro, a very low concentration of rFVIIa (0.24 microg/ml) induced thrombin generation in FXI-deficient plasma quite similarly to 1.9 microg/ml (a concentration that is achieved in patients with haemophilia A and inhibitor after infusion of 80 microg/kg). Based on this finding, a protocol was designed for four patients with severe FXI deficiency and an inhibitor or immunoglobulin A deficiency who underwent five major surgical procedures. This included administration of tranexamic acid from two hours before surgery until seven to 14 days after, and single infusion of low-dose rFVIIa. No excessive bleeding or thrombosis were observed. In conclusion, a single low dose of rFVIIa and tranexamic acid secure normal haemostasis in patients with severe FXI deficiency who can not receive blood products.

Diagnosis of thrombotic thrombocytopenic purpura based on modulation by patient plasma of normal platelet adhesion under flow condition

Summary. We have designed a simple test for the early diagnosis and treatment monitoring of thrombotic thrombocytopenic purpura (TTP). We examined plasma from 24 TTP patients and normal plasma using a cone and plate(let) analyser (CPA). Test plasma was mixed with citrated normal whole blood (group O) and subjected to flow at a shear rate of 1800/s. Mixing normal plasma (12·5, 25, 50 or 75 µl) with heterologous normal whole blood (final volume of 200 µl) resulted in a decrease of surface coverage (SC, maximally by 63%) and, to a lesser extent, of average size (AS, maximally by 37%) due to dilution of the blood sample. In contrast, mixing the same quantities of acute TTP plasma with normal blood yielded an increase in both SC (up to 125%) and AS (up to 130%). Increased SC and/or AS were detected in all 15 patients in acute phase and in three out of 14 patients in remission. Following repeated plasmapheresis, the enhanced platelet deposition in five patients with acute TTP returned to almost normal patterns. Mixing plasma from patients with other thrombocytopenic conditions in this way resulted in a decrease in both SC and AS, and did not differ from control subjects. In conclusion, the CPA is a simple and specific laboratory test that can be used for the diagnosis and monitoring of plasma exchange therapy in TTP.

Lower doses of rFVIIa therapy are safe and effective for surgical interventions in patients with severe FXI deficiency and inhibitors

Summary.  Severe FXI deficiency is a rare injury‐related bleeding disorder. In patients with FXI inhibitors, surgeries may be treated using recombinant activated factor VII; however, treatment safety is a major concern and the best dosing regimen as well as mode of administration is still to be defined. We describe four patients with severe factor XI deficiency and inhibitors to FXI, undergoing eight (four major) surgical procedures treated with continuous infusion of rFVIIa. Following acute MI that evolved after surgery of our first patient, all other patients were treated with low‐dose bolus rFVIIa followed by low‐dose continuous infusion of rFVIIa. Haemostasis was successfully achieved and no further thrombotic complications occurred. To support our clinical results ex‐vivo thromboelastography studies were performed, demonstrating the differences of clot formation and lysis between patients with FXI deficiency and healthy controls and suggesting that low‐dose rFVIIa corrects coagulation similarly to high‐dose rFVIIa in FXI deficiency. Recombinant FVIIa at low doses may effectively induce haemostasis and seems to be a safe treatment mode in patients with FXI deficiency and inhibitors undergoing surgeries.

Point of care testing in children undergoing cardiopulmonary bypass.

Excessive hemorrhage is a major complication after cardiac surgery requiring cardiopulmonary bypass (CPB). The aim of this study was to define the potential role of the cone and platelet analyzer (CPA) and the rotating thromboelastogram (ROTEM) point of care tests in children undergoing CPB.

Role of agr (RNAIII) in Staphylococcus aureus adherence to fibrinogen, fibronectin, platelets and endothelial cells under static and flow conditions

In the present study, the adherence of Staphylococcus aureus (strain 83254) and its RNAIII mutant (WA400) to immobilised fibrinogen and fibronectin, and to human endothelial cells (EC), was studied. [3H]Thymidine-labelled bacteria in stationary phase were incubated on the test surfaces for 20 min under static or flow (200/s) conditions. The results showed: (a) increased adherence of the RNAIII mutant to fibrinogen under static conditions, and decreased adherence of the mutant to fibronectin and EC under both static and flow conditions compared with the parental strain; (b) stronger ability of the mutant compared with the parental strain to induce platelet aggregation in suspension; (c) greater adherence of the parental strain and the mutant to EC pre-treated with platelet-rich plasma compared with platelet-poor plasma, and to EC pre-treated with platelet-poor plasma compared with control; (d) increased adherence of S. aureus to EC pre-treated with PMA-activated platelets and decreased adherence to EC pre-treated with tirofiban, a platelet glycoprotein IIb-IlIa inhibitor, which paralleled with increased adherence of PMA-activated platelets and decreased adherence of glycoprotein IIb-IIIa-blocked platelets to EC in the absence of bacteria; and (e) adherence of the mutant was more sensitive to pre-treatment of EC with plasma and PMA-activated platelets. In conclusion, RNAIII down-regulates S. aureus adherence to fibrinogen under static condition and up-regulates S. aureus adherence to fibronectin and EC under both static and flow conditions. The potentiating role of activated platelets in the presence of plasma in S. aureus adherence to EC is down-regulated by RNAIII, probably due to down-regulation of adherence to fibrinogen, the important plasma protein bridging S. aureus, platelets and EC.

Factor XI Deficiency Protects Against Atherogenesis in Apolipoprotein E/Factor XI Double Knockout Mice

Objective— Atherosclerosis and atherothrombosis are still major causes of mortality in the Western world, even after the widespread use of cholesterol-lowering medications. Recently, an association between local thrombin generation and atherosclerotic burden has been reported. Here, we studied the role of factor XI (FXI) deficiency in the process of atherosclerosis in mice. Approach and Results— Apolipoprotein E/FXI double knockout mice, created for the first time in our laboratory. There was no difference in cholesterol levels or lipoprotein profiles between apolipoprotein E knockout and double knockout mice. Nevertheless, in 24-week-old double knockout mice, the atherosclerotic lesion area in the aortic sinus was reduced by 32% (P=0.004) in comparison with apolipoprotein E knockout mice. In 42-week-old double knockout mice, FXI deficiency inhibited atherosclerosis progression significantly in the aortic sinus (25% reduction, P=0.024) and in the aortic arch (49% reduction, P=0.028), with a prominent reduction of macrophage infiltration in the atherosclerotic lesions. Conclusions— FXI deprivation was shown to slow down atherogenesis in mice. The results suggest that the development of atherosclerosis can be prevented by targeting FXI.