Increased procoagulant cell-derived microparticles (C-MP) in splenectomized patients with ITP.

BACKGROUND: Splenectomy is frequently employed for therapeutic and diagnostic purposes in various clinical disorders. However its long-term safety is not well elucidated. Although risk of infection by encapsulated organisms is widely recognized, less well-known are risks of thrombosis and cardiovascular disease. METHODS: We investigated levels of cell-derived microparticles (C-MP) in 23 splenectomized ITP (ITP-S) and 53 unsplenectomized ITP patients (ITP-nS). Assay of C-MP derived from platelets (PMP), leukocytes (LMP), red cells (RMP) and endothelial cells (EMP) were performed by flow cytometry. Coagulation parameters included PT, aPTT and activities of FVIII, IX and XI. Results of all measures were compared between the two groups, ITP-S vs ITP-nS. RESULTS: Levels of all C-MP were higher in ITP-S than ITP-nS but only RMP and LMP reached statistical significance (p = 0.0035 and p < 0.0001, respectively). The aPTT was significantly shorter in ITP-S (p = 0.029). Interestingly, correlation analysis revealed that RMP, but not other C-MP, were associated with shortening of aPTT (p = 0.024) as well as with increased activities of factors VIII (p = 0.023), IX (p = 0.021) and XI (p = 0.0089). CONCLUSIONS: RMP and LMP were significantly elevated in splenectomized compared to non-splenectomized ITP patients. This suggests that the spleen functions to clear procoagulant C-MP, and that elevation of C-MP might contribute to increased risk of thrombosis, progression of atherosclerosis and cardiovascular disease following splenectomy.

Microparticle-mediated thrombin generation assay: increased activity in patients with recurrent thrombosis.

BACKGROUND: Circulating cell-derived microparticles (MP) are important players in thrombogenesis, attributed in part to tissue factor (TF) carried on them. We developed MP-mediated thrombin generation assay (TGA) and measured a series of patients with thrombosis (TBS) and normal controls (NC). METHODS: MP were isolated from plasma of 66 patients with TBS and 34 NC. The MP were resuspended in normal pooled particle-free plasma (PFP) containing corn trypsin inhibitor (to inhibit contact pathway). MP mediated TGA yields three parameters: lag time, peak and rate. This method is not influenced by anticoagulant therapy. Of the TBS patients, 41 had only a single thrombosis (S-TBS) and 25 had recurrences (R-TBS) within a 5-year period. In parallel, MP were quantitated by flow cytometry, and cell origin was determined: endothelial cells (EMP), leukocytes (LMP), red cells (RMP) and platelets (PMP). RESULTS: MP from all TBS patients exhibited higher thrombin generation than NC by all three TGA parameters. R-TBS had significantly greater TGA values than S-TBS, reflected in higher peak and rate, and shorter lag time. MP numbers were also higher in TBS vs. NC, for all MP subtypes, and were significantly higher in R-TBS than S-TBS (except LMP). All MP levels correlated with thrombin generation (P < 0.0001), most closely between PMP and peak (R = 0.47) and rate (R = 0.43). CONCLUSIONS: MP-mediated TGA is a novel way to assess functional procoagulant activity of MP. Enhanced MP-mediated TGA was demonstrated in TBS patients, and significantly higher activity in R-TBS. These findings support a major role of MP in thrombogenesis.

Endothelial microparticles induce formation of platelet aggregates via a von Willebrand factor/ristocetin dependent pathway, rendering them resistant to dissociation.

Endothelial microparticles (EMP) released from activated or apoptotic endothelial cells (EC) are emerging as useful markers for detection of EC dysfunction. Our recent observation that EMP carry von Willebrand factor (vWf) led us to investigate their interaction with platelets. EMP were incubated with normal washed platelets in the presence or absence of ristocetin, then platelet aggregates were measured by flow cytometry. In the absence of ristocetin, negligible EMP conjugated with platelets (< 5%) but in the presence of ristocetin (1 mg mL(-1)), EMP induced up to 95% of platelets to aggregate. EMP-platelet interaction was 80% blocked by anti-CD42b, or by 0.1 microm filtration to remove EMP. Platelet aggregates induced by normal plasma or high molecular weight vWf (Humate-P) dissociated 50% within 15-25 min following 1:20 dilution. In contrast, aggregates formed with EMP persisted two- to threefold longer with the same treatment, indicating greater stability. A similar degree of prolongation of dissociation was observed using plasma from thrombotic thrombocytopenic purpura (TTP) patients compared with normal plasma. Addition of EMP to plasma from severe von Willebrand disease restored his ristocetin-induced platelet aggregation. Multimer analysis of vWf on EMP showed unusually large vWf (ULvWf). In summary, EMP carries ULvWf multimers, promote platelet aggregates, and increase the stability of the aggregates thus formed.

Elevated plasma endothelial microparticles in multiple sclerosis.

OBJECTIVE: To assess endothelial dysfunction in patients with MS and to investigate whether plasma from patients with MS induces endothelial cell dysfunction in vitro. BACKGROUND: Endothelial cell dysfunction may contribute to the pathogenesis of MS. Elevations of soluble adhesion molecules intracellular adhesion molecule, vascular cell adhesion molecule, and platelet-endothelial cell adhesion molecule-1 (CD31) have been reported as markers of blood-brain barrier (BBB) damage in MS, but direct assay of endothelium has been difficult. Endothelial cells release microparticles < approximately 1.5 microm (EMP) during activation or apoptosis. The authors developed a flow cytometric assay of EMP and studied EMP as markers of endothelial damage in MS. METHODS: Platelet-poor plasma (PPP) from 50 patients with MS (30 in exacerbation and 20 in remission) and 48 controls were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD31 and anti-CD51 (vitronectin receptor) antibodies, and two classes of EMP (CD31+ and CD51+) were assayed by flow cytometry. For in vitro studies, patients' plasma was added to the microvascular endothelial cell (MVEC) culture and release of CD31+ and CD51+ EMP were measured in the supernatant. RESULTS: Plasma from patients in exacerbation had 2.85-fold elevation of CD31+ EMP as compared with healthy controls, returning to near control value during remission. The CD31+ EMP concentration showed a positive association with gadolinium enhancement in patients with MS. In contrast, CD51+ EMP remained elevated in both exacerbation and remission. This suggests that CD31+ EMP is a marker of acute injury, whereas CD51+ EMP reflects chronic injury of endothelium. MS plasma induced release of both CD31+ and CD51+ EMP from MVEC culture in vitro. CONCLUSION: Endothelial dysfunction is evident during exacerbation of MS, evidenced by shedding of EMP expressing PECAM-1 (CD31). The in vitro data indicate contribution of one or more plasma factors in endothelial dysfunction of MS.

Elevated endothelial microparticles in thrombotic thrombocytopenic purpura: findings from brain and renal microvascular cell culture and patients with active disease.

Endothelial injury is believed to be a key initiating event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), leading to platelet activation and formation of platelet-rich thrombi in microvasculature. However, the nature of endothelial injury in TTP is poorly defined and clinical assays to rapidly and reliably monitor endothelial damage are not readily available. Using flow cytometry, we measured endothelial microparticles (EMPs) generated from cultured renal and brain microvascular endothelial cells (MVECs) during activation and apoptosis, and evaluated the effect of TTP plasma on them. EMPs were measured using positivity for monoclonal antibodies (mAbs) CD31 and CD51, and their procoagulant activity was assessed using a Russell viper venom assay. Both cell lines generated procoagulant EMPs when cultured with inducers of activation (tumour necrosis factor alpha; TNF-alpha) or apoptosis (mitomycin C). TTP plasma induced a five- to sixfold increase of EMP generation and a two- to threefold increase of procoagulant activity in cultured brain and renal MVECs. TTP plasma induced a threefold and 13-fold increase of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, respectively, on renal MVECs. Procoagulant activity tended to parallel EMP numbers. The effect of TTP plasma on cell viability was similar to that of TNF-alpha, implying that it induced activation rather than apoptosis. Control plasma and idiopathic thrombocytopenic purpura (ITP) plasma had little effect. In the clinical study, EMP assay of blood from acute TTP patients showed levels markedly elevated compared with normal controls, but values returned to normal in remission. In conclusion, TTP plasma activated and induced injury to MVECs in culture, judged by production of EMP and expression of activation markers. Released procoagulant EMP may play a role in the pathogenesis of TTP. Assay of EMP may be a useful marker of disease activity and endothelial injury in TTP and possibly other thrombotic disorders.

A flow cytometric assay of platelet activation marker P-selectin (CD62P) distinguishes heparin-induced thrombocytopenia (HIT) from HIT with thrombosis (HITT).

Heparin induced thrombocytopenia (HIT) is a well-known complication of heparin administration but usually resolves upon discontinuation without sequelae. However, a small proportion of HIT patients develop thrombosis associated with HIT, designated as HITT, which is often life-threatening and may lead to gangrene and amputations. Existing laboratory methods of confirming HIT/HITT do not distinguish between HIT and HITT. We report a flow cytometric assay of platelet activation marker CD62P to distinguish the effects of addition of HIT vs. HITT plasma to normal blood. Briefly, normal whole blood was incubated with platelet-poor plasma from 12 patients with HITT, 30 with HIT, and 65 controls, in presence and absence of heparin, and expression of CD62P was assayed by flow cytometry. When the ratios of fluorescent intensity of CD62P with heparin divided by that without heparin were compared, HITT plasma induced significantly higher ratios than HIT plasma (HITT ratios approximately 2.5 vs. HIT ratios approximately 1.2; p <0.001). Eleven of 12 HITT patients were positive by this test but only 5 of 30 HIT patients were positive (p <0.0005). In a case of HIT with silent thrombosis, this assay gave a positive results prior to clinically evident thrombosis. In conclusion, this method distinguishes HITT from HIT and may be clinically useful in the detection of HITT, allowing early intervention for preventing catastrophic thrombosis.

Anti-CD36 autoantibodies in thrombotic thrombocytopenic purpura and other thrombotic disorders: identification of an 85 kD form of CD36 as a target antigen.

The presence of anti-CD36 antibodies in plasma of patients with thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), and heparin-induced thrombocytopenia without/with thrombosis (HIT/HITT) has been examined by immunoblots, and a monoclonal antibody capture assay, the platelet-associated IgG characterization assay (PAICA). Results with PAICA showed that 73% (8/11) of patients with TTP were positive, and 71% (10/14) by immunoblots. With ITP, 20% (6/30) were positive by PAICA and 19% (3/16) by immunoblots; HIT, 30% (3/10) were positive by PAICA and 60% (6/10) by immunoblot; HITT, 50% (2/4) by PAICA and 100% (4/4) by immunoblot. Purification of CD36 by fast protein liquid chromatography (FPLC) from Triton X-100 extracts of normal platelet membranes resulted in the isolation of two different forms: the classic 88 kD form, and a second, lighter 85 kD form. Our data indicated that the patients' plasma autoantibodies reacted strongly with the 85 kD form. Conventional monoclonal and polyclonal antisera produced to the 88 kD form reacted strongly with the 88 kD form but weakly with the 85 kD form. These results confirm the possible importance of anti-CD36 antibodies in the pathophysiology of TTP and other thrombocytopenias and demonstrate the presence of a previously unrecognized target antigen for these antibodies.

Platelet activation in Alzheimer disease.

BACKGROUND: In light of recent reports of diminished platelet serotonin concentration and increased plasma serotonin levels in patients with Alzheimer disease (AD), we hypothesized that a state of heightened platelet activation might be present in AD. OBJECTIVE: To compare baseline activation of unstimulated platelets in patients with AD with that in control subjects. PATIENTS AND METHODS: Flow cytometry was used to measure platelet activation in 91 patients with probable AD and 40 age-matched control subjects. Groups were compared for percentage of circulating platelet aggregates, expression of CD62p, formation of leukocyte-platelet complexes, and presence of circulating platelet microparticles, controlling for effects of demographic, clinical, physiological, and logistical factors. RESULTS: Multiple analysis of covariance on ranked data revealed a 39.5% increase in percentage of platelet aggregates (P=.0001), a 59.3% increase in expression of CD62p (P=.001), and a 53.3% increase in leukocyte-platelet complexes (P=.0001) in the group with AD but no differences in the number of platelet microparticles, overall platelet count, plasma fibrinogen level, or plasma platelet factor 3. Activation was weakly correlated with sex, but was independent of age, severity of disease, duration of disease, depression, agitation, and family history of dementia. CONCLUSIONS: Platelets of patients with AD exhibit greater unstimulated activation than those of controls. Potential causes of such activation include possible stimulation of platelets by damaged cerebral endothelial cells or platelet activation induced by membrane abnormalities previously reported to be present in platelets of patients with AD. In light of recent evidence that platelets are the principal source of both amyloid precursor protein and beta-amyloid peptide in human blood, it is possible that AD platelet activation may reflect or even contribute to the pathogenesis of the disease.

Thrombotic thrombocytopenic purpura plasma enhances platelet-leucocyte interaction in vitro.

In thrombotic thrombocytopenic purpura (TTP), intravascular platelet aggregation and formation of platelet-rich thrombi impair the microcirculation. TTP plasma has been shown to induce aggregation of normal platelets in vitro. The present study investigates the formation of activated platelet aggregates (aPAg) induced by TTP plasma, with particular attention to their binding to leucocytes (LPAg). Results were compared with the effects of plasmas from normal controls (CTL) and from patients with immune thrombocytopenic purpura (ITP) or thrombosis (THR). Following addition of test plasma to normal whole blood (WB), aPAg and LPAg were assayed by flow cytometry using mAbs against CD41 (platelet marker), CD62p (platelet activation marker) and CD45 (pan-leucocyte marker), Compared to control plasma, TTP plasma was more potent than ITP or THR plasma in increasing aPAg: only TTP plasma significantly promoted leucocyte binding to give increased LPAg. Prior removal of neutrophils (PMN) from WB by beads coated with anti-CD15 mAb largely prevented formation of aPAg and LPAg. However, TTP plasma added to normal platelet-rich plasma significantly increased aPAg, which suggested possible hindrance of aPAg formation by erythrocytes and other leucocytes in PMN-depleted blood. We concluded that TTP plasma was most potent in the induction of aPAg and unique in promoting LPAg formation in WB. Neutrophils, and not other leucocytes, appear to be essential for LPAg formation. Enhanced PMN-platelet interaction in the microcirculation may facilitate platelet adhesion to vessel walls and promote the formation of platelet-rich microthrombi in TTP.

Platelet aggregates as markers of platelet activation: characterization of flow cytometric method suitable for clinical applications.

The present paper describes a flow cytometric method for assay of platelet aggregates (PAg) in blood. This method combines and simplifies previously reported techniques, simultaneously enumerating PAg formed upon platelet activation, their expression of activation marker CD62P (P-selectin), and their content of bound leukocytes (LPAg). The sensitivity of this method to low levels of agonists (ADP, collagen) is compared to conventional aggregometry and some features of platelet-leukocyte interaction are explored. The results were: (1) ADP or collagen induced a dose-dependent increase in PAg number and corresponding decline in free platelets. The ED50 for ADP (0.15 microM) and for collagen (0.2 microg/mL) was about 1/20 the ED50 found by aggregometry, indicating 20-fold greater sensitivity. (2) At higher concentrations, the fraction of PAg with bound leukocytes (LPAg) increased to 60-70%. This rise correlated with PAg size and CD62P expression, but not with the number of PAg formed. (3) The response of whole blood (WBD) to agonists was qualitatively different from that of platelet-rich plasma (PRP): in WBD the population of CD62P+ PAg was much higher than in PRP and the population of CD62P+ free platelets was much lower. This implies that leukocytes rapidly recruit activated platelets. (4) Desmopressin (DDAVP) at 5 nmol/L induced a significant rise in activated (CD62P+) PAg and platelets, even though no effect of DDAVP could be detected by conventional aggregometry; this further confirms that DDAVP acts directly on platelets. (5) Plasma samples from TTP patients induced a rise in PAg when added to normal PRP, though little or no effect could be detected by aggregometry. In summary, the flow cytometric method described here appears useful for detecting low levels of platelet activation and provides information on platelet leukocyte interaction, potentially important in identifying and differentiating thrombogenic states. Since it is rapid and economical, it is well suited for clinical implementation.

Platelet microparticles and calcium homeostasis in acute coronary ischemias.

UNLABELLED: Elevation of free cytoplasmic calcium is the common pathway of platelet activation, leading to shape change, shedding of platelet microparticles (PMP), aggregation, and secretion of internal granules, including expression of CD62p on the surface. Platelet activation is well documented in unstable angina (UA) and acute myocardial infarction (MI). We investigated the following markers of platelet activation in 55 patients undergoing coronary angiography for suspected CAD: free cytoplasmic calcium, [Ca2+]cyt, PMP, CD62p expression, and platelet/leukocyte (P/L) interaction. [Ca2+]cyt was measured by Fluo-3 and the other measurements were by flow cytometry. Patients were classified into three groups: unstable angina (UA, n = 11), recent myocardial infarction (MI, n = 11), and patient controls (CTL, n = 33). Blood was drawn before infusion of heparin through femoral lines at the time of catheterizaton for assays. ( RESULTS: (1) PMP values were significantly higher in both UA and MI than in CTL, P < 0.05. There was no difference between UA and MI. (2) P/L interaction was significantly elevated only in UA, P < 0.05. (3) CD62p expression on free platelets did not differ significantly between any of the three groups. (4) The resting [Ca2+]cyt, thrombin-induced Ca2+ influx, and release of Ca2+ from internal stores were all significantly higher in platelets from the combined patient group (UA + MI) than in the patient control group, P < 0.001 CONCLUSIONS: Results on calcium hemostasis and PMP were significantly different in patients with acute coronary syndromes than those with stable angina or no coronary ischemia; this may reflect underlying pathophysiology of acute coronary ischemia. P/L interaction was higher only in the UA group, suggesting a role of leukocytes in UA.

Danazol for paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal stem-cell disorder in which blood cells lack complement inhibiting membrane proteins, and become susceptible to complement-mediated injury, leading to chronic intravascular hemolysis and pancytopenia. Glucocorticoids have been a mainstay of therapy. For patients refractory to glucocorticoids and requiring blood transfusions, an alternative therapy is needed. We studied danazol therapy in 5 patients refractory to other treatments. Four of the 5 benefited, showing rise in hematocrit and eventual cessation of transfusion requirements. Remissions lasted > or =2 years in 3 and 10 years in 1 patient. Danazol was well-tolerated without serious side effects. Danazol appears to be a good alternative treatment in PNH.

Activated platelet aggregates in thrombotic thromboctyopenic purpura: decrease with plasma infusions and normalization in remission.

Circulating activated platelet aggregates (aPA) were assayed by flow cytometry employing mAb alpha-CD62p in eight patients with thrombotic thrombocytopenic purpura (TTP). Elevation of aPA was observed in all patients in active stages of TTP; aPA normalized in remission. Plasma infusions with plasmapheresis decreased aPA in responding patients. The rise and fall of aPA preceded relapses and improvements, respectively. These changes were seen prior to the traditional indicators, LDH, haematocrit, and platelet count. Incubation of plasma from TTP patients with normal whole blood induced formation of aPA; this effect was significantly greater than that of plasmas from ITP patient controls (P < 0.01), suggesting the presence of an aPA-promoting factor in TTP plasma. Parallel experiments using a platelet aggregometer failed to detect effect of TTP plasma on normal blood. In summary, aPA appear to be a marker of disease activity, rising with relapse, falling with plasma therapy, and normalizing in remission. The flow cytometric assay of aPA is more sensitive than aggregometry in detecting the putative aPA-promoting factor in TTP.

Danazol relieves refractory pruritus associated with myeloproliferative disorders and other diseases.

Severe pruritus is frequently associated with myeloproliferative and other systemic illnesses, and often fails to respond to conventional measures. We used danazol (Danocrine), a synthetic attenuated androgen, in the treatment of severe pruritus refractory to conventional therapy. Eight patients had myeloproliferative disorders (MPD), seven had autoimmune disorders, and seven had skin diseases. Danazol at 400-800 mg/day was administered, and previous medications were tapered off. When itching was controlled with danazol alone, the dosage was reduced or discontinued, and resumed if itching recurred. Clinical responses were graded, and side effects were monitored. Overall, in 12 of 22 patients refractory to other measures, itching was controlled with danazol alone. In 10 patients itching returned when danazol was discontinued or dosage was continued for up to 5 years in responders. No serious side effects were observed. Our experience indicates that danazol is a good alternative for patients with severe pruritus associated with myeloproliferative and other systemic disorders.

Platelet factor 3 in plasma fractions: its relation to microparticle size and thromboses.

Platelet factor 3 (PF3) was assayed by Russell's viper venom (RVV) in three plasma fractions, platelet-rich plasma (PRP), platelet poor plasma (PPP), and 0.1 microns particle-filtered plasma (PFP), in 42 healthy controls, 34 patients with recent cerebrovascular accidents (CVA) and 28 with recent ischemic events from coronary artery disease (CAD). Platelet microparticles (PMP) were assayed in PPP by flow cytometry. Relative to controls, the RVV clotting times were shortened in all three plasma fractions in both patient groups, p < 0.001. PMP were also elevated in both patient groups, p < 0.001. Linear regression analysis showed that the RVV times of PPP are inversely correlated with PMP, p < 0.005, in patient groups but not in controls. There was no correlation of RVV time with PT, APTT or FIB. After converting RVV times to units of PF3 activity, it could be shown that only about 1/4 of the total PF3 activity was contributed by platelets. The major contribution to the PF3 activity in controls was from microparticles < 0.1 microns but in patients was due mainly to microparticles > 0.1 microns. The RVV time was superior to routine coagulation tests in discriminating thrombotic patients from healthy controls.

Danazol distribution in plasma and cell membranes as related to altered cell properties: implications for mechanism.

Concentrations of danazol in patient plasma and red blood cells (RBC) were assayed over a 6-month period in 75 patients on danazol therapy using a high-pressure liquid chromatography (HPLC) method more reliable than previous radioimmunoassay (RIA) methods. It was found that plasma danazol rose regularly for 15 days after the beginning of treatment, reaching a steady state plateau of 175 +/- 76 ng/ml in 20 patients on normal dose, and less for lower dose schedules. After stopping danazol, concentrations declined to near zero in a similar time frame. RBC concentrations on a packed volume basis were similar to plasma levels. However, the membrane ghosts of RBC contained about 50% of the total RBC danazol, implying about 100-fold higher concentration in membranes than in plasma. Similar distributions were obtained in vitro with both RBC and platelets, and were confirmed by 14-C-labeled danazol. These findings tend to support the hypothesis that the benefits of danazol in immune disorders may be attributable in part to its intercalation in the lipid bilayer of the plasma membrane, altering antigen/receptor expression to modulate immune reactions. This hypothesis was first suggested when it was observed that the RBC of patients on danazol therapy showed morphological changes and increased resistance to osmotic lysis. It was later shown that danazol in vitro reduces binding of autoantibodies, and protects against complement-mediated lysis, suggesting direct action of danazol on the membranes. This hypothesis is discussed, and danazol's effect in protecting against complement-mediated lysis is described.

Desmopressin (DDAVP) acts on platelets to generate platelet microparticles and enhanced procoagulant activity.

Desmopressin (DDAVP), an analog of vasopressin (AVP), has wide clinical application as an anti-hemorrhagic (AH) agent. DDAVP in vivo releases vWF from endothelial cells but is reported to have little action on platelets. However, DDAVP is often used to improve hemostasis in platelet dysfunctions. We examined the effect of DDAVP on platelet microparticle (PMP) formation and procoagulant activity in vitro using platelets from normal volunteers and in vivo in six patients receiving DDAVP therapy. In the former, platelets were incubated with DDAVP (0.5 to 25 nM) and PMP released were stained with FITC-labeled MAb alpha-GP IIb/IIIa for flow cytometry. Procoagulant activity was measured in a clot-based assay using Russel's viper venom (RVV) calibrated with cephalin. A mean increase of 2-3 fold was observed in both PMP and procoagulant activity. Parallel to these observations was a dose-dependent rise in organelle-associated Ca2+. The assays were also performed on six patients prior to and at one hour after infusion of DDAVP, and similar but lesser effects were observed. We conclude that DDAVP acts on platelets in vitro, and that these effects may contribute to the hemostatic action of DDAVP in platelet dysfunctions in vivo.