Robert Rosenstein

Reduced tissue factor (thromboplastin) activity in von Willebrand's disease

Abstract Identification of tissue factor in cultures of human skin fibroblasts permitted assessment of tissue factor activity in patients with bleeding disorders. Tissue factor activity was studied by means of a one-stage assay patterned after the prothrombin time and a two-stage test in which activation of factor X by tissue factor in the presence of factor VII was assessed. Tissue factor activity observed in cultured skin fibroblasts from five subjects with hemophilia A and B was not significantly different from that observed in pooled fibroblasts from normal controls. By contrast, tissue factor activity in cultured skin fibroblasts from four subjects with von Willebrands disease was significantly lower than that in the control. It is postulated that the observed tissue factor defect may be related to the pathogenesis of von Willebrands disease.

Substitution of tetramethylbenzidine for benzidine in cyanide analyses

Benzidine (B, p-diaminodiphenyl) has been used in analytical laboratories for many years in assays for plasma hemoglobin [l], inorganic cyanide, and thiocyanate ]2,3]. The chemistry involved in the two assays for the inorganic anions is the same, but quite different from the hemoglobin assay. In the classical assay for plasma hemoglobin, the hemoglobin functions as a pseudoperoxidase and B, in the presence of hydrogen peroxide, is converted to a quinoid chromophore, B blue, and eventually to other quinoid forms [I]. In the assay for cyanide and thiocyanate, both are converted to cyanogen bromide by excess elemental bromine. Arsenous acid is used to destroy the excess bromine. Cyanogen bromide is then reacted with pyridine to generate glutaconic aldehyde which in turn reacts with B to form a Schiff base chromophore [4]. Because B is an established carcinogen, there is increasing reluctance to permit its use for these analytical applications and severe restrictions have been placed on its production [S]. A non-carcinogenic derivative of B, 3,3’,5,5’-tetramethylben~dine (TMB), has been successfully substituted in the plasma hemoglobin assay without loss of sensitivity, precision, or accuracy [6]. Here we show that the same is true for the assay of cyanide and thiocyanate. This new technique has been applied to measurements of cyanide released in vitro from sodium nitroprusside (SNP) during incubations with human red blood cells, platelets, and plasma.

The diagnostic value of the aspirin tolerance test

Abstract The aspirin tolerance test was systematically applied to 56 individuals evaluated in our coagulation laboratory and its diagnostic value assessed. The test was only utilized when the pre-aspirin bleeding time did not exceed 8 min, a time below which 95% of our normals fell. When plotted on probability paper both pre- and postaspirin bleeding times appeared to be log normally distributed with the only readily apparent difference being a shift in the mean. Nine of the tested group were diagnosed as having (or probably having) platelet-related bleeding disorders. However, none of these individuals showed a post-aspirin bleeding time that differentiated them from the rest of the population. The failure of the aspirin tolerance test to eliminate the need for other diagnostic procedures leads us to recommend that this procedure not be used as a preliminary diagnostic ‘screening’ test.

TISSUE FACTOR: A VITAMIN K-DEPENDENT CLOTTING FACTOR?

Tissues from many organs have long been known to possess a potent initiator of blood coagulation that is not present in p1asma.l This coagulant, commonly referred to as tissue thromboplastin, is perhaps best known as a laboratory reagent used to perform the one-stage prothrombin time test. The traditional mystique surrounding the supposed nonspecificity of “thromboplastin” has given way to increasingly precise biochemical characterization of this substance1 and the utility of this term has declined. Reference to the tissue coagulant as “tissue factor” (TF, factor HI), rather than thromboplastin, i s now more appropriate since it is known to be a specific glycoprotein. TF apoprotein, while lacking coagulant activity itself, becomes a potent coagulant when combined with phospholipid, especially when present in a membrane configuration. TF exerts its coagulant effect upon combining with an activating plasma coagulation factor VII. In so doing, fibrin formation is initiated by way of the so-called extrinsic (or TF-activated) pathway of blood coagu1ation.l It is not the purpose of this review to present an exhaustive and critical review of the literature on TF. Rather, certain aspects of the cellular biology of TF will be reviewed in order to place in context the recent finding that TF coagulant activity is reduced by warfarin administration. This raises the possibility that TF may be a vitamin K-dependent coagulation factor.

Effect of temperature on short-term preservation of platelets for in vitro tests.

We have evaluated the effect on the response of citrated platelet-rich plasma to aggregating agents of storage at 4 degrees C versus room temperature (21 degrees C) for 2 and 4 h after venipuncture. While there were small decreases in some responses to epinephrine, ADP and collagen attributable to 21 degrees C storage, only in the case of ristocetin-induced aggregation was a profound difference noted. Platelets stored at 4 degrees C for 4 h showed no significant change in response to ristocetin. In contrast, those stored at room temperature showed a marked decrease. This change is not likely to be attributable to a change of pH, although pH rose less with storage at 4 degrees C than at 21 degrees C. It is recommended that PRP for in vitro testing be stored at 4 degrees C.

Drug-related changes in platelet shape in whole blood

The need to understand the role platelets play in both hemostasis and thrombosis has served as the impetus for development of numerous in vitro tests of platelet behavior Cll. While many of these appear useful for the detec&ofatelet defects in disorders of hemostasis, their relevance to the detection of alterations in in vivo platelet behavior, either pathologic [2-41 or drug induced i-51, appears unclear. -Indeed, while platelet inhibitory drugs have profound effects in vitro, their clinical effects are either more modest or unpredictable C61. This problem has prompted the search for tests which might provide a more meaningful measure of in vivo platelet activity. --