A preliminary study of platelet activation after embolization of marrow contents.

OBJECTIVES: Intravasation of bone marrow contents into venous circulation and pulmonary embolization after intramedullary nailing may be coupled with the activation of coagulation and fibrinolytic cascades. The objective of this study was to assess hemostatic response to pulmonary extravasated marrow contents. We hypothesize that activation of platelet activity and the coagulation cascade may occur after embolization of marrow contents in an experimental animal model of intramedullary nailing. METHODS: Fifteen New Zealand white male rabbits were randomly assigned to control or fat embolism (FE) groups. In the FE group (n = 8), femurs were surgically instrumented with retrograde intramedullary nails and pressurized with bone cement. In the control group (n = 7), a sham knee incision was made that was immediately closed without drilling, reaming, or pressurization. Fibrinogen, D-dimer latex screen assay, 1 stage prothrombin time, and activated partial thromboplastin time were analyzed. RESULTS: As the main platelet activation indicators, the marker Annexin-V percent binding increased in the FE group at 2 hours (P = 0.04) and 4 hours (P = 0.04), and the marker CD62P percent expression increased in the FE group at 2 hours (P = 0.04). CONCLUSIONS: This preliminary study showed that pressurization of marrow and intravasation of fat and marrow products cause activation of platelets and the coagulation cascade, with or without tissue trauma. This may be relevant to the treatment of multiply injured patients with prior respiratory and coagulation abnormalities. A future larger study may be needed.

Porcine factor VIII in the treatment of high-titre inhibitor patients.

Development of an inhibitor against factor VIII (FVIII) is an important complication of haemophilia. It occurs in approximately 25-30% of patients with haemophilia A [1]. FVIII inhibitors may also occur as autoantibodies. The latter occur in non-haemophiliacs and, although rare (occurring in approximately one per million of the population), are frequently associated with life-threatening bleeding. Inhibitors are considered low level if they are < 5 Bethesda Units (BU) or high level if they are > 10 BU. The former usually remain low and rarely give anamnestic response, the latter do so frequently. Despite various approaches to their management, the presence of inhibitors remains a major cause of morbidity and mortality. The effectiveness of porcine FVIII (pFVIII) in treating patients with both auto- and alloantibodies to FVIII has been well demonstrated when adequate circulating levels of FVIII are obtained [2--10]. However, pFVIII therapy may give rise to antibodies to the pFVIII and the utility of this treatment in the presence of high levels of porcine antibody is less well recognized and understood. Nonetheless, pFVIII under these circumstances may be useful in a select group of patients where management is difficult.

Platelet activation and hypercoagulability following treatment with porcine factor VIII (HYATE:C).

Activation of platelets and coagulation in vivo was studied in nine patients with hemophilia A and inhibitors to human Factor VIII, prior to and following treatment with porcine Factor VIII (PFVIII; HYATE:C). In addition, six hemophiliac patients were similarly studied after treatment with recombinant Factor VIII (rFVIII). Platelet activation was also examined in vitro using porcine von Willebrand factor (PvWF)-enriched and PvWF-depleted fractions obtained by fractionation of PFVIII. Coagulation was assessed by measuring the concentrations of plasma prothrombin fragment 1+2 concentrations (prothrombinase generation) and Factor Xa-ATIII. Patients treated with PFVIII had significantly increased numbers of circulating platelets expressing CD62 and CD63 (markers of platelet activation) and annexin V (marker of platelet procoagulant activity) compared to patients treated with rFVIII; the former patients also demonstrated an increase in plasma coagulability after therapy. In in vitro experiments it was observed that the platelet-activating and procoagulant capacity of PFVIII resided in the PvWF-enriched fraction, and the same was true for the plasma hypercoagulability following exposure of platelets to PFVIII. These results support the hypothesis that PFVIII-induced platelet activation provides a mechanism for enhancing hemostasis, separate from, and additional to, that due to increased circulating Factor VIII, and it is due to residual PvWF in the PFVIII preparation.

Porcine factor VIII provides clinical benefit to patients with high levels of inhibitors to human and porcine factor VIII.

The development of an inhibitor against Factor VIII is an important complication of haemophilia and occurs in approximately 31% of patients [1]. Despite various approaches to their management, the presence of these inhibitors remains a major cause of morbidity and mortality. Inhibitors may be low level [<5 Bethesda Units (BU)] or high level [>10 BU]. Low-level inhibitors usually remain low and do not respond to administered factor VIII with a rise of the inhibitor titre; high-level inhibitors, in contrast, are characterized by a rapid rise in titre following treatment with factor VIII. Modalities of treatment of acute bleeding episodes in patients with inhibitors include 'overcoming' the inhibitor with very large doses of factor VIII, 'bypassing' the inhibitor blockade with products such as activated prothrombin complex concentrates or recombinant factor VIIa, 'removing' the inhibitor by plasmapheresis or immunoabsorption and 'repressing' it with immunosuppressive drugs and immune tolerance induction. An alternative approach is to use a porcine factor VIII concentrate which does not cross-react with the human factor VIII inhibitor. Following treatment with porcine factor VIII, functional factor VIII can be detected and the therapeutic levels correlate with cessation of bleeding. The use of porcine factor VIII may, however, result in the development of antiporcine factor VIII antibodies, limiting its use. Experience in patients with high-titre inhibitors indicates that porcine factor VIII therapy could be used in the presence of factor VIII inhibitors [2-11], including inhibitors to porcine factor VIII [5,7], and that haemostasis may be obtained in the absence of detectable levels of circulating factor VIII [7,11].

Characterization of platelet glycoproteins and platelet/endothelial cell antibodies in patients with thrombotic thrombocytopenic purpura.

Platelets and sera from 12 patients with thrombotic thrombocytopenic purpura (TTP) and 12 healthy normal control subjects were examined. As determined by quantitative flow cytometry, prior to plasma exchange therapy platelet surface glycoprotein (GP) Ib levels were similar in TTP patients and normal controls (mean 20 188 and 20 226 molecules/platelet, respectively). Platelets from patients with TTP did, however, have significantly reduced levels of GPIIb/IIIa prior to plasmapheresis (mean 36 348 v 52 505 molecules/platelet in controls; P = 0.0004) and of GPIV (mean 13 321 v 26 212 molecules/platelet in controls; P = 0.0002). An increase in activated platelets, as determined by CD62 expression, was observed in 82% of patients. Increased platelet-associated immunoglobulins and/or complement was also seen in approximately 60% of the patients. In general, with return of platelet counts to normal levels following seven plasmaphereses, the above abnormalities were reversed, although often not to normal levels. Western blot analysis indicated the presence of antibodies reactive to platelet GPIV (88 kD) in 70% of pretreatment sera from patients with TTP; a similar band was observed in 80% of patient sera against microvascular endothelial cells. Immunofluorescence microscopic examination indicated the presence of antibody in pretreatment sera from patients with TTP to microvascular (73%) and large vessel (36%) endothelial cells. As measured by an indirect flow cytometric assay, pretreatment sera from 55% of patients with TTP were reactive with large vessel endothelial cells and 100% reacted with microvascular endothelial cells; reactivity was significantly greater against the microvascular endothelial cells (P = 0.0048) and was reduced following plasma exchange therapy. These results indicate abnormalities in platelet glycoprotein expression in TTP and suggest that anti-platelet and anti-endothelial cell antibodies play a role in the thrombocytopenia and vasculitis characteristic of this disorder.

Plasma urokinase antigen and plasminogen activator inhibitor-1 antigen levels predict angiographic coronary restenosis.

BACKGROUND: The fibrinolytic system is intimately involved in several processes that contribute to restenosis, including clot dissolution, cell migration, and tissue remodeling. However, the role of the individual activators (urokinase [uPA] and tissue plasminogen [tPA] activators) and inhibitors (plasminogen activator inhibitor [PAI-1]) of the fibrinolytic system in maintaining patency after coronary artery angioplasty and stenting is unclear. METHODS AND RESULTS: We prospectively studied 159 patients with stable angina who underwent successful elective angioplasty (n=110) or stenting (n=49) of de novo native coronary artery lesions. Plasma samples were drawn at baseline (before angioplasty) and serially after angioplasty (immediately afterward and 6 hours, 24 hours, 3 days, 7 days, 1 month, 3 months, and 6 months afterward). Antigen and activity assays were performed for uPA, tPA, and PAI-1. Follow-up quantitative coronary angiography was performed in 92% of eligible patients. The overall angiographic restenosis rate (diameter stenosis >50%) was 31% (37% in PTCA patients, 17% in stented patients). At all time periods, including baseline, uPA antigen levels were significantly higher and PAI-1 antigen levels were significantly lower in patients with restenosis. Restenosis rates for patients in the upper tertile of baseline uPA antigen levels were 2-fold higher than for those in the lower 2 tertiles (46% versus 24% and 22%, respectively; P<0.004). In a stepwise regression multivariate analysis, obstruction diameter after the procedure and uPA antigen were significant predictors of follow-up diameter stenosis. CONCLUSIONS: Plasma uPA antigen levels and PAI-1 antigen levels identify patients at increased risk for restenosis after percutaneous coronary revascularization.

Directions for research in autoimmune thrombocytopenic purpura (ITP).

All attendees participated in a round-table discussion regarding directions for research in autoimmune thrombocytopenic purpura (ITP). Suggested areas for study were grouped into five main areas: (i) improved classification of ITP identifying subsets of patients with differing clinical syndromes and response to treatment, and those more likely to have serious bleeding manifestations; identification of patients with reduced thrombopoiesis was emphasized; (ii) studies aimed at elucidating the aetiology and pathophysiology of ITP, with emphasis on distinctions between acute and chronic ITP and between patients responsive or refractory to therapy; these studies focused on measures of humoral and cellular immune dysregulation; (iii) studies of platelet function in ITP, with the intent of defining these abnormalities and correlating them with the clinical manifestations of the disease; (iv) new approaches to treatment, particularly of refractory patients; and (v) a miscellaneous group, which included development of an ITP registry, evaluation of the "burden" of disease, investigation of mood changes in ITP, etc. The discussion was not intended to be all-inclusive, but focused on the content of other talks in this symposium. It is hoped that some of these suggestions will be further developed for investigation in multicentre co-operative studies to improve the diagnosis, understanding and treatment of ITP.

Platelet activation induced by porcine factor VIII (Hyate:C).

We studied the effects of porcine factor VIII (P-FVIII; Hyate:C) and other coagulation products employed in the management of patients with hemophilia A, on platelet activation in vitro. Exposure of normal resting platelets to P-FVIII resulted in platelet activation, as manifested by increased expression of the platelet surface activation markers CD62, CD63, and activated-GPIIbIIIa, and by activation-induced modulation of expression of normal platelet membrane glycoproteins CD41, CD42, and CD36. In contrast, platelet activation was not observed after exposure of the platelets to human FVIII, FEIBA, recombinant FVIIa, or cryosupernatant plasma. As with thrombin, exposure of platelets to P-FVIII resulted in the generation of platelet microparticles, an effect not seen not with the other products. In contrast to the characteristic reduction in expression in the number of CD42 molecules detected on thrombin-activated platelets, P-FVIII-stimulated platelets showed a small increase in CD42 expression. In contrast to thrombin, P-FVIII did not cause platelet dense granule release. The results indicate that therapeutic P-FVIII activates platelets, likely in ways that are different from the platelet activation seen with thrombin. The observed platelet activation and microparticle generation may provide a "hypercoagulable" mechanism for hemostasis with P-FVIII therapy separate from, and additional to, that due to increased circulating FVIII levels.

Platelet cold agglutinins: a flow cytometric analysis.

Spontaneous EDTA-independent cold platelet agglutination is a rare phenomenon that produces pseudothrombocytopenia when blood samples are analyzed in automated cell counters. We report a case of platelet cold agglutinins and an analysis by flow cytometry. A 49 year old woman presented with abnormal vaginal bleed secondary to uterine fibroids. Platelet clumping was observed in blood samples taken in EDTA-, heparin- and citrate-containing tubes. In flow cytometric tests, patient serum agglutinated 16% of normal platelets at 22 degrees C, and 7% of platelets after incubation at 37 degrees C; in contrast, 3% and < 1% of platelets were agglutinated at 22 and 37 degrees C, respectively, after incubation with normal serum. Minimal agglutination (< 10%) was observed with patient serum at a titre of 1:5 or at temperatures > 30 degrees C. After incubation at 4 degrees C, IgM antibody and C3 were increased on the patient's platelets; no significant amount of IgM or C3 was detected on normal platelets. The specificity of the platelet cold agglutinin was determined by competitive inhibition by monoclonal anti-CD41(GPIIbIIIa). Before the addition of monoclonal antibody, patient's serum agglutinated 16% of normal platelets at 22 degrees C; after addition of anti-CD41 only 2% of the platelets were agglutinated. This blocking effect was not observed with anti-CD42. The patient's platelets functioned normally as determined by CD62 and CD63 expression in response to thrombin, normal platelet aggregation in response to collagen, ADP, and ristocetin, and a normal template bleeding time. In summary, platelet agglutination by a platelet cold agglutinin was quantitated by flow cytometry, the responsible antibody was characterized as a low titre IgM with minimal activity > 30 degrees C, and competitive binding studies supported the GPIIbIIIa complex as the binding site for the antibody. Since the antibody did not affect platelet function, we believe that these patients will not suffer complications from their platelet cold agglutinin, but it could pose a problem under circumstances such as cardiac surgery with hypothermia.

Effect of psyllium in hypercholesterolemia at two monounsaturated fatty acid intakes.

We performed two studies to determine whether the lipid-lowering effect of viscous soluble fiber was modified by monounsaturated fatty acid (MUFA). First, psyllium (1.4 g/MJ) was compared with wheat bran (control) in 1-mo metabolic diets by using a randomized crossover design (n = 32 hyperlipidemic subjects). The background diet contained approximately 6% of energy as MUFA (20% of total fat). The second study (n = 27 hyperlipidemic subjects) was similar to the first but the background diet contained approximately 12% MUFA (29% of total fat) because of the addition of canola oil. At both fat intakes, psyllium resulted in significant reductions in total, low-density-lipoprotein (LDL), and high-density-lipoprotein (HDL) cholesterol compared with the wheat bran control. For the psyllium diet at 6% compared with 12% MUFA, the decreases in LDL cholesterol were 12.3 +/- 1.5% (P < 0.001) and 15.3 +/- 2.4% (P < 0.001), respectively. With the higher-MUFA diet triacylglycerol fell significantly over the control phase (16.6 +/- 5.5%, P = 0.006) and the ratio of LDL to HDL cholesterol fell significantly over the psyllium phase (7.3 +/- 2.8%, P = 0.015). Psyllium and MUFA intakes were negatively related to the percentage change in the ratio of LDL to HDL cholesterol (r = -0.34, P = 0.019 and r = -0.44, P = 0.002, respectively). Chenodeoxycholate synthesis rate increased (30 +/- 13%, P = 0.038) with the psyllium diet in the 12 subjects in whom this was assessed. We conclude that psyllium lowered LDL- and HDL-cholesterol concentrations similarly at both MUFA intakes. However, there may be some advantage in combining soluble fiber and MUFA to reduce the ratio of LDL to HDL cholesterol.

Hypofibrinolysis in patients with hypercoagulability: the roles of urokinase and of plasminogen activator inhibitor.

The prevalence of abnormalities of fibrinolysis in patients with venous thromboembolism is as yet unknown. Defined abnormalities include congenital dysfunction and deficiency of plasminogen, and probably impaired plasminogen activation secondary to elevated levels of plasminogen activator inhibitor type 1 (PAI-1) or to impaired release of tissue plasminogen activator (tPA). In this preliminary study, we analyzed plasma samples from 21 patients for whom an investigation for possible thrombophilia was requested. Twenty of the patients had venous thromboembolism, and one had arterial thrombosis at an early age. Two patients had deficiency of protein C or protein S, but no other recognized biochemical disturbances related to thrombophilia were identified. Patient samples and plasma from 25 normal controls were assayed for tPA activity, PAI-1 activity, and urokinase (uPA) activity and antigen. tPA activity and antigen were not significantly different in patients than in controls. PAI-1 activity was significantly greater in patients (P < 0.0001). uPA activity was not different in the two groups. However, uPA antigen was significantly reduced in patients compared to controls (P = 0.001). These data suggest that hypofibrinolysis leading to a risk of thrombosis may be caused not only by elevated PAI-1 activity but also by reduced total uPA concentration.

White cell depletion of red cell and pooled random-donor platelet concentrates by filtration and residual lymphocyte subset analysis.

This study comparing the relative white cell (WBC)-depleting efficiency of single and double filtration used two filters and new, sensitive, and reliable methods for performing WBC counts on WBC-depleted blood products. A single filtration of red cell (RBC) concentrates with a cotton-wool filter reduced WBC content by 98.64 percent, but the range of residual WBCs was wide, and many filtered units still contained more than the theoretical immunizing dose of 5 to 10 x 10(6) WBCs. A second filtration, however, always produced RBC units that had less than 5 x 10(6) WBCs. Although the degree of WBC depletion observed after a single filtration of a 6-unit pool of random-donor platelet concentrates was greater with a polyester filter than with the cotton-wool filter (98.92 vs. 98.14% reduction, respectively, when mean prefiltration WBC count was less than 600 x 10(6], in both cases, 25 percent of filtered products still contained greater than 5 x 10(6) WBCs; a second filtration (with the cotton-wool filter), however, produced units that were always below the immunizing dose. All double-filtered platelet concentrates had less than 10(6), and one-half had less than 10(4) residual WBCs. Platelet loss was similar with both filters (+/- 16% loss with one filtration). The effectiveness of the filters in producing products that were WBC-depleted below the immunizing dose was dependent on the prefiltration WBC content (but not on the age of the units), and it may be worthwhile to employ methods to ensure that total prefiltration WBC count of the product is less than 400 x 10(6).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute leukemia evolving from multiple myeloma and co-expressing myeloid and plasma cell antigens.

This report describes the development of acute myeloblastic leukemia in a patient after long-term alkylator therapy for multiple myeloma. Despite chromosome deletions -5, -7, the patient lacked the histochemistry and clinical findings characteristic of therapy-induced leukemia. In double-labeled surface marker studies by flow cytometry, the leukemic blast cells co-expressed myeloid and plasma cell surface markers. The findings may support the hypothesis of a single stem cell abnormality's being responsible for both the malignant plasma cells and the myeloid leukemic cells.

Two-year evaluation of clinical and laboratory variables of immune function in 117 hemophiliacs seropositive or seronegative for HIV-1.

Fifty-nine HIV-1 antibody positive and 58 antibody negative hemophiliacs were evaluated over a 2 year study period to gain insight into the natural history and prognosis of HIV-1 disease in members of this risk group. Mean CD4 (Leu 3+) cell counts calculated at 6 month intervals decreased gradually in seropositive patients (from 403 to 311/microliters) whereas CD8 (Leu 2+) counts remained stable but above the normal range. CD4 cell counts correlated closely with advancing CDC clinical stage; CD8 numbers showed no such association, but were markedly lower in the six patients with overt AIDS. Serum P24 antigenemia was associated with low CD4 cell counts and with advanced clinical stage (58% of antigenemic and 14% of non-antigenemic seropositive patients were in stage IV). In addition to CD4 cell counts, significant reductions in Leu 11+ natural killer cell (NK) subsets and in Leu 3 + 8 - cells occurred in seropositive patients over the study period; Leu 2 + DR + cells increased significantly. When expressed as a percentage of lymphocytes, the reduction in Leu 19 + NK cells was also significant, as were the increases in Leu 4 + DR + cells and Leu 12 + 8 + B cells. In summary, declining CD4 cell numbers and percentages are valuable markers of progressive HIV-1 disease in hemophiliacs, but may not always accurately reflect the degree of disease activity. Progressive changes in additional variables such as serum P24 antigen, and numbers and percentages of NK cell subsets and (as AIDS supervenes) CD8 cell numbers, may allow more precise monitoring of HIV-1 disease. This will, in turn, facilitate the design of optimal individualized strategies for therapeutic intervention.

A cost-effectiveness evaluation of platelet crossmatching and HLA matching in the management of alloimmunized thrombocytopenic patients.

Effective platelet support for alloimmunized refractory thrombocytopenic patients may be provided by several potential strategies, the most common being HLA-matched single-donor platelets or crossmatch-compatible, pooled random- or single-donor platelets. This study used a detailed economic analysis to compare the cost-effectiveness of several techniques for platelet crossmatching and that of HLA-matched single-donor platelets. The crossmatch methods evaluated were a microlymphocytotoxicity test (LCT), an immunofluorescence technique (PSIFT), a radioactive antiglobulin test (PRAT), and an enzyme-linked immunosorbent assay (ELISA). The analysis was based on the need to support 100 refractory patients with acute leukemia with a presumed requirement of 500 transfusions. The relative costs for a successful crossmatch were: PRAT less than LCT less than LCT + PRAT less than PSIFT less than ELISA. In the comparison of the crossmatch methods, an increase in costs was generally associated with an increase in the number of successful transfusion episodes. However, decreasing marginal gains were seen. The HLA-matched single-donor platelets were relatively cost-inefficient in comparison to the crossmatch-compatible platelets. A theoretic sequence of tests for cost-effective provision of optimal platelet support in refractory patients was evaluated. Such considerations of cost are important in the selection of an optimal program for the management of alloimmunized refractory thrombocytopenic patients.

Carrier detection in hemophilia A: ABO blood group, multiple measurements, and application of logistic discrimination.

In healthy 20- to 50-year-old women, the ABO blood group has a significant effect on levels of von Willebrand factor (VWF:Ag, formerly VIIIR:Ag) and on factor VIII activity (F.VIII:C). However, there is no significant effect of ABO group or subject age on the ratio log e(F.VIII:C/VWF:Ag). Multiple measurements of the "ratio" on possible carriers of hemophilia A may be combined with pedigree information using logistic discrimination to yield final risk assessment. To reduce misclassification of carriers as normal women, a lower limit, specified by the logistic model, is set on the logistic carrier probabilities. In this study, the proportion of blood group A for a population of obligate carriers was significantly higher than that expected for the general population (60% vs. 42%); for a population of control women it was lower than expected (22.5 vs. 42%). The effect for the carriers came primarily from daughters of affected fathers, as 81.3% were of blood group A. These observations indicate that a "universal" discriminant should be applied with caution.

The endothelial cell and the factor VIII bypassing activity of prothrombin complex concentrate.

Some classical hemophiliacs have a paradoxical hemostatic response to prothrombin complex concentrate (PCC). We hypothesized that vascular endothelial cells (EC) may contribute to this "factor VIII bypassing activity". When PCC were incubated with suspensions or monolayer cultures of EC, they acquired the ability to partially bypass the defect of factor VIII deficient plasma. This factor VIII bypassing activity distributed with EC and not with the supernatant PCC, and was not a general property of intravascular cells. The effect of PCC was even more dramatic on fixed EC monolayers, which became procoagulant after incubation with PCC. The time courses of association and dissociation of the PCC-derived factor VIII bypassing activity of fixed and viable EC monolayers were both rapid. We conclude that EC may provide a privileged site for sequestration of constituents of PCC which express coagulant activity and which bypass the abnormality of factor VIII deficient plasma.

Random donor platelet crossmatching: comparison of four platelet antibody detection methods.

The standard lymphocytotoxicity assay (LCT), a biotin-avidin enzyme immunoassay (ELISA), platelet suspension immunofluorescence test (PSIFT), and platelet radioactive antiglobulin test (PRAT) were examined in prospective crossmatching for selection of compatible random donor platelets for refractory patients. One hundred seven episodes of pooled random donor platelet transfusions were evaluated in 26 patients. There was good reproducibility of results by individual techniques. Concordance of results by the different methods was 40-60%. One-hour and 24 hr posttransfusion corrected count increments (CCI) were compared as parameters for assessing success or failure of the transfusion. Using a rank scoring system, the relative efficiency of predictiveness for all transfusions was PRAT greater than LCT greater than PSIFT greater than ELISA. Combination of PRAT and LCT afforded the best predictability and sensitivity was higher than for either PRAT or LCT alone (93 vs. 79 and 62%, respectively). Mean posttransfusion CCI (x 10(9)/L) following PRAT-compatible platelets was 13.9 +/- 12.7 at 1 hr and 7.3 +/- 6.9 at 24 hr; following PRAT-incompatible platelets, 5.7 +/- 7.8 (1 hr) and 2.1 +/- 4.1 (24 hr). Results were similar for LCT-tested platelets. A radioimmunofiltration modification of the PRAT developed and used in selected cases was simple, fast, efficient, and inexpensive. The study indicated that the techniques evaluated are practical and feasible for routine use in the provision of compatible random donor platelets to the refractory patient who has no other cause for increased platelet destruction.