Morris A. Blajchman

The contribution of the haematocrit to thrombocytopenic bleeding in experimental animals

Summary Clinical studies in anaemic uraemic patients have shown that increasing the haematocrit with either red blood cell (RBC) transfusions or erythropoietin corrects the prolonged bleeding time (BT) often seen in such individuals. In this present study we evaluated experimentally the effect of the haematocrit on the BT using a microvascular BT technique in New Zealand White rabbits. The correlation between haematocrit and BT was studied in both normal and thrombocytopenic rabbits. In non‐thrombocytopenic animals the microvascular BT varied inversely with the haematocrit (r=‐0.799); animals with haematocrit levels above 35% having significantly shorter BTs than animals with haematocrit values lower than 35% (P < 0.001). To assess the role of the haematocrit on the BT in thrombocytopenic animals, thrombocytopenia was induced by a combination of γ‐irradiation and heterologous platelet antiserum. Such experiments showed that anaemic rabbits had significantly longer BTs than non‐anaemic animals with a similar degree of thrombocytopenia (P=0.0001). These data thus provide evidence that anaemia contributes significantly to the prolonged BT in both thrombocytopenic and non‐thrombocytopenic rabbits, and that RBC transfusions are capable of shortening the BT in thrombocytopenic anaemic animals. While results obtained from animal models cannot necessarily be extrapolated to the clinical situation, the fact that haematocrit influences the BT must be taken into account in the assessment of anaemic patients, particularly those who may have an associated haemostatic disorder.

Experimental animal model of refractoriness to donor platelets: the effect of plasma removal and the extent of white cell reduction on allogeneic alloimmunization.

Platelet transfusion effectiveness may be limited in multiply transfused patients by the development of the refractory state. White cell (WBC)‐reduction filters with variable efficiency (1–3 log10 reduction) are available and have been shown to be effective in reducing the incidence of platelet alloimmunization. However, the threshold number of WBCs below which alloimmunization would no longer occur is yet to be determined. A previously established animal model was used to examine the relative efficiency of second‐ and third‐ generation filters in reducing the frequency of refractoriness to allogeneic platelets. In this model, California Black rabbits are used as blood donors and New Zealand White rabbits as transfusion recipients. Eight weekly transfusions of either second‐generation or third‐generation WBC‐reduced blood resulted in no difference between the two groups in mean platelet survival and rate of refractoriness to allogeneic platelets. To evaluate the possible incremental benefit of removing supernatant plasma to prevent platelet refractoriness, experiments were performed in which groups of animals were given transfusion(s) with red cell suspensions that had been WBC‐reduced or both plasma‐depleted and WBC‐reduced. A significantly lower rate of allogeneic platelet refractoriness was seen in the rabbits that received WBC‐reduced and plasma‐depleted red cells than in those that received red cells that had been WBC‐reduced only. These data provide evidence that the combined use of plasma depletion and WBC reduction can decrease still further the frequency of refractoriness produced by allogeneic blood transfusions.

Platelet Transfusion‐Induced Serratia Marcescens Sepsis due to Vacuum Tube Contamination

Three instances of Serratia marcescens septicemia in two patients following infusion of platelet concentrates stored at 22 C, and the isolation of the organism from one unit of a platelet concentrate, led to a study to determine the possible sources of such contamination. Cultures of the available blood products, derived from the same blood donations used to prepare the suspect platelet concentrates, yielded Serratia marcescens from two units of cryoprecipitate and from one unit of red blood cells. All other available blood products were sterile. Serratia marcescens was isolated in considerable numbers from 82 per cent of the vacuum tubes from one manufacturers lot in use in the transfusion center at the time of the septic episodes. Six other lots of vacuum tubes prepared by the same manufacturer in use at the same time were sterile. The organism was not found in samples from other equipment, materials or personnel involved in the preparation of the blood products. Simulation of the blood collection technique using vacuum tubes from the contaminated lot, when filled from an in‐line needle as used following the blood collection procedure, gave contamination of the primary pack with Serratia marcescens in five of the six experiments attempted. The contaminated vacuum tubes were thus considered the most likely source of contamination of the platelet concentrates.

Age‐related changes in factor VII proteolysis in vivo

Previous studies have reported that pre‐operative plasmas of patients over the age of 40 years who developed post‐operative deep vein thrombosis (DVT) had approximately twice the amount of proteolysed factor VII found in plasmas of patients in whom prophylaxis with heparin or low Mr heparin was successful. These and other studies also reported higher concentrations of thrombin–antithrombin III in pre‐ and post‐operative plasmas of patients who developed post‐operative thrombosis than in plasmas of patients in whom prophylaxis was successful. Whether the extent of factor VII proteolysis seen in the patients who developed post‐operative DVT is related to the severity of their disease or age is not known. This report investigated age‐related changes in the concentrations of total factor VII protein, factor VII zymogen, factor VIIa, tissue factor pathway inhibitor, thrombin–antithrombin III, and prothrombin fragment 1+2 in normal plasmas and the relationships between these parameters. With the exception of thrombin–antithrombin III, statistically significant increases in the concentrations of these parameters with age were found. Additionally, the differences between the concentrations of total factor VII protein and factor VII zymogen, an index factor VII proteolysis in vivo, were statistically significant only for individuals over age 40. Using linear regression analysis, a significant correlation was found to exist between the concentrations of plasma factor VIIa and prothrombin fragment 1+2. Since factor VIIa–tissue factor probably initiates coagulation in vivo, we hypothesize that the elevated plasma factor VIIa (reflecting a less tightly regulated tissue factor activity and therefore increased thrombin production in vivo) accounts for the high risk for post‐operative thrombosis seen in individuals over the age of 40.

The inhibition of the anticoagulant activity of heparin by platelets, brain phospholipids, and tissue factor

Summary. Platelets and phosphilipids have been shown to protect factor Xa from inhibition by the heparin‐antithrombin III complex. The studies reported herein investigated the effects of gel filtered platelets, activated platelets, brain phospholipids (cephalin), and brain tissue factor on the inactivation of thrombin and factor Xa by the heparin‐antithrombin III complex. In addition, the relative anticoagulant effects of heparin on the extrinsic and intrinsic coagulation pathways were investigated. Our results suggest that gel filtered platelets, activated platelets, cephalin and tissue factor protect thrombin, as well as factor Xa, from inactivation by the heparin‐antithrombin III complex. Tissue factor had the greatest anti‐heparin activity. Activated platelets, gel filtered platelets, cephalin and tissue factor did not alter the protease‐antithrombin III reaction rates measured in the absence of heparin. These observations are consistent with the hypothesis that platelets, brain phospholipids, and tissue factor, in the presence of calcium, partition heparin from antithrombin III, and thus prevent full expression of the antithrombin III‐dependent anticoagulant activity of heparin.

In vitro evaluation of prestorage pooled leukoreduced whole blood–derived platelets stored for up to 7 days

BACKGROUND: Advantages to storing whole blood–derived platelets (PLTs) as a pool for 7 days would include operational efficiencies and facilitation of bacterial testing and pathogen inactivation. The in vitro quality of pre‐storage pooled PLTs stored for up to 7 days was assessed.

Assessing the effectiveness of whole blood–derived platelets stored as a pool: a randomized block noninferiority trial

BACKGROUND: Prestorage pooling of whole blood–derived platelets (PLTs) would simplify bacterial detection. This study evaluated the in vivo effect of the prestorage pooling of PLTs stored for up to 5 days, by assessing the corrected count increment (CCI) 18 to 24 hours after transfusion of the product.

Plasma Anticoagulant Mechanisms of Heparin, Heparan Sulfate, and Dermatan Sulfate

The relationship between two anticoagulant actions of glycosaminoglycans (GAGs), namely the catalysis of thrombin inhibition (assessed by thrombin-antithrombin-III and thrombin-heparin-cofactor-II formation) and the inhibition of prothrombin activation, was explored by comparing the effects of heparin, heparan sulfate, and dermatan sulfate on the two reactions in plasma. Heparan sulfate and dermatan sulfate were also resulfated in vitro to yield products with sulfate/carboxylate ratios similar to those of heparin. Their effects on thrombin inhibition and the activation of prothrombin were also determined. The catalytic efficiency of the five GAGs on thrombin inhibition and their inhibitory effects on prothrombin activation decreased in the following order: heparin; resulfated dermatan sulfate; resulfated heparan sulfate; heparan sulfate = dermatan sulfate. These results suggest that the catalytic efficiency of a glycosaminoglycan on thrombin inhibition translates to its inhibitory effect on prothrombin activation, since catalysis of thrombin inhibition results in the inhibition of the thrombin-dependent positive feedback reactions of coagulation which facilitate prothrombinase formation.

Mechanisms for Inhibition of the Generation of Thrombin Activity by Sulfated Polysaccharidesa

Three mechanisms by which sulfated polysaccharides act as anticoagulants and possibly as antithrombotic agents have been described. These are the two heparin cofactor-dependent mechanisms involving the catalysis of the inhibition of various proteases of coagulation by either antithrombin III or heparin cofactor II. The third is a heparin cofactor-independent mechanism involving the inhibition of formation of prothrombinase and tenase complexes. Four sulfated polysaccharides previously shown to have anticoagulant and antithrombotic effects were assessed to determine which of the three mechanisms operate in the expression of their anticoagulant effects. To do this, [125I]prothrombin was added to undiluted human plasma, and the inhibition of [125I]prothrombin activation, or the catalysis of the formation of thrombin-inhibitor complexes was determined in plasma containing one of the four sulfated polysaccharides. Prothrombin activation was demonstrated by the formation of [125I]prothrombin fragment 1.2 and [125I]thrombin. The effect of the thrombin-specific inhibitor, D-Phe-L-Pro-L-ArgCH2Cl (PPACK), on prothrombin activation was also investigated to determine the role of thrombin-dependent feedback reactions on efficient prothrombin activation. Use of PPACK with sulfated polysaccharides also facilitated estimation of the role of the heparin cofactor-independent effects of sulfated polysaccharides on prothrombin activation. Three concentrations of each of the sulfated polysaccharides were used: 0.66, 6.6, and 66 micrograms/ml of plasma. PPACK (1.0 X 10(-6)M) completely inhibited both intrinsic and extrinsic prothrombin activation. The inhibition of prothrombin activation caused by PPACK was abolished when thrombin was added to the plasma before PPACK. These observations indicate that the presence of trace thrombin activity is critical for efficient prothrombin activation by both the intrinsic and extrinsic pathways. All three concentrations of standard heparin completely inhibited the intrinsic activation of prothrombin. This inhibition was only partially abolished when thrombin was added to the plasma before heparin, indicating that heparin inhibits prothrombin activation both by catalyzing the inhibition of thrombin activity and by a heparin cofactor-independent mechanism. Heparan sulfate did not inhibit intrinsic prothrombin activation but catalyzed the inhibition of the thrombin generated by the formation of thrombin-antithrombin III complex. Dematan sulfate inhibited intrinsic prothrombin activation only at the highest concentration. At the two lower concentrations, dermatan sulfate catalyzed formation of thrombin-heparin cofactor II.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of blood transfusions on experimental pulmonary metastases in mice

We examined the effect of allogeneic blood transfusions (BT) on pulmonary metastases in a mouse model. Recipient (C57B1/6J) mice were transfused with either saline, syngeneic blood or allogeneic (Balb/c) blood on two occasions, days 0 and 3. One week after the last transfusion, recipient mice were injected intravenously with varying numbers of methylcholanthrene‐induced fibrosarcoma cells. Twenty days later the number of pleural nodules was counted as an index of pulmonary metastasis. The data demonstrate that the inoculation of 2.5 times 105 or 1 times 105 tumor cells resulted in significantly higher numbers of pulmonary metastases in mice that received allogeneic BT than the mice that received syngeneic blood or saline. In contrast, allogeneic BT caused significant inhibition of pulmonary metastases in mice that received 3.5 times 105 tumor cells. The data suggest that the immunomodulatory (stimulatory or inhibitory) effect of BT is dependent on the numbers of tumor cells inoculated. It is likely that the conflicting reports in the literature on the effects of BT on tumor growth may be due to inoculation of different numbers of tumor cells. These results have an important bearing in understanding the effect of allogeneic BT on tumor growth both in experimental animals and in cancer patients.