Thrombotic Events in Homozygotes with a Proven or Highly Probable Arg304Gln Factor VII Mutation (FVII Padua)1): Only Limited Replacement Therapy is Needed in Case of Surgery

Objective: To investigate the prevalence of thrombotic events among patients with proven or highly probable homozygosis for the Arg304Gln (Factor VII Padua) defect or compound heterozygosis containing the Arg304Gln mutation. Methods: Homozygotes and compound heterozygotes proven by molecular studies to have the Arg304Gln mutation were gathered from personal files and from two PubMed searches. In addition, patients with probable homozygosis on the basis of clotting tests (discrepancies among Factor VII activity levels according to the tissue thromboplastin used) were also gathered. Results: 30 proven homozygotes and 17 probable ones were gathered together with 8 compound heterozygotes. In the latter use, the associated mutation was Cys135Arg (twice), Gly180Arg, Arg304Trp, Arg315Trp, His348Gln, Gly365Cys. The prevalence of venous thrombotic events was 16.6, 11.8 and 11.1 percent, respectively for the three groups of patients. Heterozygotes showed no thrombotic event. The difference for proven homozygotes was statistically significant, while for the other groups only a trend was present. Conclusion: proven homozygous or compound heterozygous patients with the Arg304Gln mutation showed a higher than expected incidence of thrombotic events. The same is true for probable cases gathered only on the basis of clotting tests. These patients, because of their frequent lack of bleeding and for their relatively high prevalence of thrombosis should probably receive only limited replacement therapy in case of surgical procedures.

The development of the blood clotting initiation conceptions: from A.A. Schmidt to D.M Zubairov

The objective of the review is to cover the formation of modern understanding of molecular mechanisms of blood coagulation initiation. It was provided mainly by the research of Professor D.M. Zubairov and his colleagues. Since 1963, he has established that blood coagulation initiation is not connected to the phenomenon of vascular wall moistening and contact complex factors activation. Research of the thromboplastic activity distribution in tissue cells, blood and in the serum phospholipid microparticles allowed to conclude that blood coagulation is initiated by long-term expression of tissue factor and rapid massive alterations in cellular membranes. This was confirmed by the detection of the turned phospholipids mesophases in tissue thromboplastin preparations and heterogeneity of vitamin К-depending factors binding. Based on the results of the research, a functional conception of blood coagulation initiation by phase alteration of bilayer structure of cellular membranes to a mesomorphic structure was developed. It is caused by different agonists through receptor dependant Са 2+-mobilizing cell signal systems or by massive migration of calcium ions into the cell at its damage. An initial bioimitating non-enzymatic proteolysis vitamin of К-dependant factors and their massive enzymatic activating in the ensembles of enzymatic complexes takes place on heterophase phospholipids surface. Clotting is limited by blood and tissue macrophages, removing cells and phospholipids particles with heterophase surface from cell circulation, and also by anticoagulant factors action. Based on this conception, the researches revealed the pathogenetic of role thrombogenic micro vesicles originating form the cellular membranes transformation in the development of disseminated intravascular coagulation syndrome, myocardial infarction, leucosis, autoimmune and infectious diseases. Finding out the basic concepts of blood coagulation initiation mechanism puts D.M. Zubairov in one row with scientists, pawning the bases of modern biology and medicine.

Increased Sensitivity of Prothrombin Time Using Recombinant Tissue Thromboplastin Reagents: Implications for Plasma Transfusion.

Acquired coagulopathies are often treated with fresh frozen plasma (FFP) transfusion. Standards for the tranfusion of FFP vary, but are often based on coagulation tests, especially prothrombin time (PT). Previously, the PT test was performed using tissue thromboplastin (TT) reagents derived from human or animal tissue homogenates (hTT). These preparations were fairly crude and varied between manufacturers and lots. Recently, these have been replaced with recombinant TT (rTT) reagents that are more consistent. However, anecdotal reports suggest that rTT is more sensitive to mildly decreased clotting factor levels, leading to longer PTs and increases in FFP transfusion. The purpose of this study is to test the hypothesis that rTT reagents are more sensitive and to determine if thresholds for FFP transfusion should be increased. Fifty plasma samples from patients with prolonged PTs (range 13.0–27.6 seconds; normal 9.2–12.8 seconds) were randomly selected. PTs for each sample were re-measured using rTT (Innovin, Dade-Behring, Liederbach, Germany; ISI=0.91) and hTT (Thromboplastin Reagent, Helena Laboratories, Beaumont, Texas; ISI=2.07) on the same instrument (Start 4 benchtop coagulation analyzer, Diagnostica Stago, Parsippany, New Jersey). These measured PTs are compared in Fig. 1. At the lower end of the PT range (beginning at ~13 seconds), the rTT PTs are shorter, on average, than the hTT PTs. However, the average rTT PT increases at a 1.4 times greater rate than the hTT PT, such that the average PT of the two reagents is equivalent at 16.4 seconds and longer for rTT thereafter. This suggests that the rTT reagent is indeed more sensitive. A similar pattern is seen when factor II and VII levels for each sample are plotted against the corresponding PT (Fig. 2). At high factor levels, the rTT PT is lower, on average, than the hTT PT. However, as factor levels decrease, the rTT PT rises about twice as fast as the hTT PT. The PTs for the two reagents are equivalent at 66% factor II (PT=15.7) and 46% factor VII (PT=17.7). The important thresholds for risk of surgical bleeding are 40% factor II and 25% factor VII. The equivalent PTs in this study are 20.5 (rTT) and 18.1 (hTT) for 40% factor II and 22.3 (rTT) and 19.6 (hTT) for 25% factor VII. These differences are not corrected by converting PTs to the international normalized ratio (INR). Instead, INRs are higher for hTT than rTT, and the degree of difference between the two reagents is greater. The equivalent INRs are 1.90 (rTT) and 2.54 (hTT) for 40% factor II activity, and 2.05 (rTT) and 2.98 (hTT) for 25% factor VII activity. The results of this study confirm that PTs measured using rTT are more sensitive to changes in clotting factor levels than those measured using hTT, especially at clinically significant factor levels. This suggests that thresholds for the transfusion of FFP should be raised at those institutions using rTT. Fig. 1: Comparison of PTs measured Using rTT and hTT Reagents Fig. 1:. Comparison of PTs measured Using rTT and hTT Reagents Fig. 2: Correlation of Prothrombin Time and Factor Levels Fig. 2:. Correlation of Prothrombin Time and Factor Levels

Thrombophilic Patients with Migraines Have a High Incidence of Antiphospholipid Antibodies.

Migraines are associated with an increased risk of stroke and are recognized as a non-stroke neurologic complicaton in the antiphospholipid syndrome (aPls). Traditional tests for antiphospholipid antibodies (APA) include PTT assays [aPTT, dRVV, and Hexagonal phospholipid (Hex PL)], a dilute PT assay (Tissue Thromboplastin Inhibition) for lupus anticoagulants (LAC), and ELISA assays for anticardiolipin antibodies (ACA). These assays have not correlated with migraines. It is now recognized that antibodies to a phospholipid binding protein called beta-2-GPI are important in autoimmune aPls. Some patients (pts) with aPls are sero-negative, eg. negative in the routine assays, but have + anti-beta-2-GPI antibodies. Animal studies have shown an association of the latter antibodies to neurologic dysfunction. This study is a clinical retrospective medical record methodology, examining the relationship between migraines and APA results in pts referred to the Hemostasis & Thrombosis Clinic at the Institute for Transfusion Medicine from 7/1/03-7/15/05. Inclusion criteria were any pt. referred to the PI, who had a LAC screening panel and anti-beta-2-GPI testing. Exclusion criteria were any pt who did not have this testing or were + without repeat testing >2 mos later. Abstracted data included history of migraines (with or without aura), opthalmic migraines, referral reason, age, sex and APA results. Results were considered to be + (in non-anticoagulated pts), if any of the clotting assays were repeatedly +, did not correct on a mix, and shortened with phospholipid. Patients with only a + clotting assay, were taken off coumadin (OAC) and retested. Patients with + ELISA assays on OAC Rx, required an abnormal dRVV mix and/or a + Hex PL assay to be considered as + for a LAC. ELISA results were considered as +, if they were low titer or higher, e.g. ≥ mean ± 3 SD. The cohort consisted of 258 pts (69M: 189F) with a median age of 48 (range=13–85). Of these, 76 (29%) had migraines. Thirty-five/258 (13.6%) of the pts were referred for risk assessment and 10/35 (28.6%) had migraines. The remaining 223 pts were referred for clinical events: arterial or venous thrombosis [n= 133/233 (59.6%)], neurologic reasons [n=62/223 (28.2%)] and other [n=27/233 (12.1%)]. Of this group, migraines were present in 66 (29.6%). None of the 10 risk pts with migraines had + results. One of the 25 risk pts had a + anti-beta-2-GPI antibody (1/25=4.0%). Positive APA were detected in 58/233 (24.9%) pts with clinical events. Table 1 is a breakdown of the APA results vs migraines in pts with events vs those evaluated for risk issues. It appears that pts with thrombotic events with migraines have a higher incidence of +APA than those who do not have migraines. This data supports the suspected association between migraines and APA in the thrombphilic pt population. The same does not appear to be true in pts (without a thrombotic event) undergoing risk assessment. Incidence of Positive APA in Patients with regard to Migraine Status Group Total Pos Total Neg P OR CI APA = LAC/ACA and/or anti-beta-2-GPI Risk-no migraine 1/25 (4%) 24/25 (96%) Risk-migraines 0/10 (0%) 10/10 (100%) 1.00 0.810 0.03–21.5 Events-ALL pts 58/233 (25%) 175/233 (75%) 0.002 11.27 1.51 to 84.2 Events-no migraine 30/157 (19%) 127/157 (81%) 0.058 5.83 0.77 to 44.22 Events-migraines 28/66 (42%) 38/66 (58%) <0.001 25.05 3.23 to 194.2