Protein C, protein S and von Willebrand factor levels correlate with bleeding symptoms: a population-based study.

Although natural anticoagulant deficiencies are the established causes of thrombosis, their roles in bleeding are not fully studied. The objective is to correlate haemostatic factors with haemorrhagic symptoms quantified by a standardized questionnaire. Adult subjects were recruited from Bangkok and nearby provinces as part of routine health surveys/checkups. The validated MCMDM-1VWD form was used to assess their bleeding symptoms. At the same time, von Willebrand factor (VWF) activity, free protein S levels and protein C activity were measured. There were 5196 individuals. The mean age was 44.3 years (range 15-99) and 41% were male subjects. The mean bleeding score was -0.28 and 95% of subjects had scores between -2 and +2. The scores were lower in female subjects than in male subjects (-0.35 vs. -0.16, P < 0.001). Bleeding scores correlated negatively with age, VWF and protein C activities (Spearman's ρ-0.258, -0.091 and -0.098, respectively, all P < 0.001), but did not significantly correlate with protein S levels. Using multivariate analysis, female gender, VWF below 100 IU dL(-1), protein C below 100 IU dL(-1) and protein S over 150 IU dL(-1) significantly related to high (≥3) bleeding scores (adjusted odds ratio 1.95, 1.83, 1.56 and 2.84, P = 0.001, 0.001, 0.039 and 0.017, respectively). These findings may suggest interacting roles of VWF and natural anticoagulants in modifying bleeding symptoms.

A simplified procedure for thromboplastin calibration--the usefulness of lyophilized plasmas assessed in a collaborative study.

The International Sensitivity Index (ISI) of thromboplastins is determined by calibration using fresh plasmas from 60 patients stabilized on oral anticoagulants and 20 healthy subjects. This procedure is demanding, particularly for those who have no easy access to patients. The alternative use of a smaller number of lyophilized plasmas has already been considered, but one important issue, the number of repeated measurements to be carried out, has never been addressed. Two commercial rabbit thromboplastins, A and B, were calibrated in 3 laboratories against CRM 149R. On each of 10 working days, prothrombin times were measured for a different set of 8 fresh plasmas and for the same set of 8 lyophilized plasmas. The ISI values for both thromboplastins were estimated by orthogonal regression on fresh and lyophilized plasmas. The between- and within-laboratory CV values of the estimated ISI were taken as measures of precision of the calibration. In addition, ISI and CV were calculated daily on cumulative results obtained with lyophilized plasmas from day 1 to day 10. The ISI values for both thromboplastins calculated with lyophilized plasmas were not significantly different from those with fresh plasmas (mean of 3 laboratories: 1.42 vs 1.48 for A and 1.22 vs 1.20 for B). The between-laboratory precision of the calibration with lyophilized plasmas was not considerably different from that with fresh plasmas (CV for 3 labs: 5.2% vs 6.8% for A and 0.9% vs 2.2% for B). The ISI estimated with lyophilized plasmas on results of day 1 were not different from those of days 2 through 10. Good within-laboratory precision of the calibration (CV around 2%) was already achieved on day 3. In conclusion, this study shows that lyophilized plasmas pooled from normals and patients on oral anticoagulants can be used as substitutes for individual fresh plasmas to simplify the existing procedure for thromboplastin calibration.

A partial factor V deficiency in anticoagulated lyophilized plasmas has been identified as a cause of the international normalized ratio discrepancy in the external quality assessment scheme.

Lyophilized normal and anticoagulated plasmas are increasingly used in External Quality Assessment schemes (EQAS) to assess the quality of performance of laboratories engaged in the control of oral anticoagulant therapy by means of the International Normalized Ratio (INR) system and for the standardization of the prothrombin time (PT). The main feature of the INR system is that the value measured for an individual plasma should be independent of the reagent used. This holds true only if the variable factors influencing the responsiveness of the reagents are the vitamin K-dependent clotting factors, the other factors being constant and within normal limits. We provide evidence that even a partial factor V (FV) deficiency (FV activity < 60 U/dl) in anticoagulated plasmas can be responsible for discrepancies between INR values as measured for a single lyophilized or fresh plasma by different reagents and that the discrepancy is proportional to and magnified by the International Sensitivity Index (ISI) of the reagent used. The greater the difference between ISI values, the wider the gap between INR values. Since FV is one of the most labile plasma clotting factors, its activity is likely to be lost during freeze-drying. Hence, we recommend a thorough control of the FV activity in commercial and home-made lyophilized plasmas before their use in EQAS and in the standardization of the PT.

Ticlopidine facilitates the deaggregation of human platelets aggregated by thrombin.

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80-90% 14C-serotonin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5x the activity of thrombin) and PGE1 (10 mumol/l) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 mumol/l) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

Lupus anticoagulant in Thai systemic lupus erythematosus patients.

In the past decade, interest in the potential clinical significance of lupus anticoagulant (LA) has grown tremendously. Recent reviews from the Western countries have found an average frequency of 34% for LA in patients with systemic lupus erythematosus (SLE). By using various laboratory procedures, namely, standard and diluted activated partial thromboplastin time, kaolin clotting time, tissue thromboplastin inhibition test and platelet neutralization test, we found the frequency of LA in 91 consecutive Thai SLE patients to be 17.5%, compared with 0.8% in the age-matched normal control population. The presence of LA was significantly associated with disease activity (p = 0.01). A statistically significant association was also observed between the presence of LA and convulsive disorders (p = 0.04), thrombocytopenia (p = 0.001) and autoimmune hemolytic anemia (p = 0.02).

Ticlopidine selectively inhibits human platelet responses to adenosine diphosphate.

Platelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrinogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPII b/III a monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their Kd were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.