Cancer-related thrombotic microangiopathy secondary to Von Willebrand factor-cleaving protease deficiency

Cancer-related thrombotic microangiopathy (TM) is a serious complication with a short-term life-threatening prognosis. This complication shares certain similarities with thrombotic thrombocytopenic purpura and hemolytic uremic syndrome, both characterized by circulating platelet aggregates containing ultralarge multimers of Von Willebrand factor (VWF). We report a case of cancer-related thrombotic microangiopathy secondary to disseminated metastatic cancer with undetectable serum Von Willebrand factor-cleaving protease activity and no evidence of serum inhibitory antibody. A concomitant decrease of Ca 19-9 level and hemolysis was observed during chemotherapy, in parallel with normalization of Von Willebrand factor-cleaving protease activity. The role of ultralarge multimers of Von Willebrand factor in platelet aggregation in the context of metastatic disease is discussed with respect to our findings in this case of cancer-related thrombotic microangiopathy.     

Advances in the pathogenesis, diagnosis and treatment of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome

The thrombotic microangiopathies are microvascular occlusive disorders characterized by hemolytic anemia caused by fragmentation of erythrocytes and thrombocytopenia due to increased platelet aggregation and thrombus formation, eventually leading to disturbed microcirculation with reduced organ perfusion. Depending on whether brain or renal lesions prevail, two different entities have been described: thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). However, not rarely the clinical distinctions between these two conditions remain questionable. Recent studies have contributed greatly to our current understanding of the molecular mechanisms leading to TTP and HUS. In this review, we briefly focus on the most important advances in the pathophysiology, diagnosis and treatment of these two thrombotic microangiopathies.     

Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets

Platelet microparticles (PMPs) are small vesicles released from blood platelets upon activation. The procoagulant activity of PMPs has been previously mainly characterized by their ability to bind coagulation factors VIII and Va in reconstructed systems. It can be supposed that PMPs can contribute to the development of thrombotic complications in the pathologic states associated with the increase of their blood concentration. In this study, we compared procoagulant properties of calcium ionophore A23187-activated platelets and PMPs using several in-vitro models of hemostasis. Surface densities of phosphatidylserine, CD61, CD62P and factor X bound per surface area unit were determined by flow cytometry. They were 2.7-, 8.4-, 4.3-, and 13-fold higher for PMPs than for activated platelets, respectively. Spatial clot growth rate (V(clot)) in the reaction-diffusion experimental model and endogenous thrombin potential (ETP) were determined in plasma, which was depleted of phospholipid cell surfaces by ultra-centrifugation and supplemented with activated platelets or PMPs at different concentrations. Both V(clot) and ETP rapidly increased with the increase of PMP or platelet concentration until saturation was reached. The plateau values of V(clot) and ETP for activated platelets and PMPs were similar. In both assays, the procoagulant activity of one PMP was almost equal to that of one activated platelet despite at least two-orders-of-magnitude difference in their surface areas. This suggests that the PMP surface is approximately 50- to 100-fold more procoagulant than the surface of activated platelets.     

Effects of ticlopidine on monoclonal anti-CD9 antibody-induced platelet aggregation and microparticle generation.

We analyzed the effects of ticlopidine on platelet aggregation and on microparticle (MP) formation when platelets were exposed to a monoclonal anti-CD9 antibody (NNKY1-19) in vitro. Even when NNKY1-19-induced platelet aggregation was completely inhibited by preincubation with anti-GPIIb/IIIa antibody or Arg-Gly-Asp-Ser, or by using washed platelets from a Glanzmann's thrombasthenia patient, the formation of MP was still observed. Prostaglandin E1 and protein kinase C antagonists (H-7 and staurosporine) inhibited both NNKY1-19-induced aggregation and MP formation. Ticlopidine or aspirin plus apyrase scarcely affected NNKY1-19-induced platelet aggregation, except to prolong the lag time. However, ticlopidine significantly inhibited MP formation (p less than 0.01). These results suggest that ticlopidine inhibits NNKY1-19-induced MP formation by a different mechanism to that of the other antagonists, and that this mechanism is unrelated to the inhibition of platelet aggregation.     

Effects of EDTA on the membrane glycoproteins IIb-IIIa complex--analysis using flow cytometry

We developed new monoclonal antibodies (NNKY1-32, NNKY2-6, NNKY2-18) that react with a determinant specific to the GPIIb-IIIa complex, and studied the distribution of GPIIb/IIIa on the platelet surface in vivo and some factors that influence the structure of GPIIb/IIIa using flow cytometry. 1. The surface of large platelets is richer in GPIIb/IIIa than that of small platelets. 2. Incubation of intact platelets with EDTA at 37 degrees C causes progressive dissociation of GPIIb-IIIa complexes, and this influence is marked in small platelets. 3. Addition of Ca2+ or Mg2+ to dissociated GPIIb-IIIa complexes causes reassociation, and additionary ADP after the cation treatment further increases the binding of monoclonal antibodies. 4. Prolonged incubation of intact platelets with EDTA at 37 degrees C induces a peculiar change of structure in the GPIIb/IIIa.     

Microparticle generation during in vitro platelet activation by anti-CD9 murine monoclonal antibodies

We used flow cytometry and two anti-CD9 murine monoclonal antibodies (NNKY1-19, MALL13) to investigate the glycoprotein composition and the potential functions of microparticles (MP) released by platelets exposed to these antibodies in vitro. NNKY1-19 produced aggregation with characteristics similar to those noted in previous reports. The action of MALL13 on platelets in platelet-rich plasma (PRP), however, differs from that of other anti-CD9 antibodies. The normal fluctuation in the MALL13-induced change in optical density disappeared when complement was present. MALL13-induced effect for platelet in PRP was not inhibited by preincubation with monoclonal anti-GPIIb/IIIa antibody, but was inhibited in washed platelets (WP). Furthermore, following MALL13 stimulation in PRP platelets, the amount of buffer LDH markedly increased and electron microscopy findings showed vacuoles appearing inside the platelets. These results suggest that MALL13 has at least two effects on platelets that differ for PRP platelets and WP. The number of MP released was increased by the addition of anti-CD9 antibodies. MP surfaces were found to be rich in CD9 protein. MALL13 stimulation lead to a significant increase in the binding of C1q and C3 to platelets and caused the production of MP to occur more rapidly than it did the exposure of fibrinogen binding sites in the presence of complement. The analysis of the relationship of MP to anti-CD9 monoclonal antibody may be useful in the investigation of the relationship between platelet function and coagulation regulation.     

Platelet agglutinating protein p37 causes platelet agglutination through its binding to membrane glycoprotein IV

A 37 kDa platelet agglutinating protein (PAP p37) has previously been shown to be present in a subset of patients with thrombotic thrombocytopenic purpura and has been purified from their plasma. Using solubilized platelet membrane proteins from normal donors, it was shown by Western blotting that 125I-p37 bound to a membrane protein of 97 kDa (red/unred). Furthermore, the same protein was identified by reverse immunoblotting in which purified p37 was electrophoresed, transferred to the nitrocellulose sheet and incubated with solubilized normal platelet membrane proteins. The complex formed between p37 and the membrane protein was identified by autoradiography using polyclonal and monoclonal (OKM5) anti-GPIV antibodies, but was not detected by polyclonal antibody to GPIIIa. Similar studies with purified platelet GPIV under both reducing and non-reducing conditions demonstrated the binding of 125I-p37. Polyclonal and monoclonal antibodies to GPIV completely inhibited the platelet agglutination induced by TTP plasma containing p37, however, normal rabbit IgG, rabbit anti-GPIIIa IgG, and murine monoclonal anti-GPIIb/IIIa (10E5) antibodies had no effect. These data indicate that platelet GPIV is the receptor site for PAP p37.     

Plasma ADAMTS13, von Willebrand factor (VWF) and VWF propeptide profiles in patients with DIC and related diseases

ADAMTS13, endothelial von Willebrand factor (VWF) and related proteins are involved in the pathogenesis of some life threatening systemic thrombotic coagulopathies. Changes of plasma ADAMTS13 activity in thrombotic thrombocytopenic purpura (TTP) is well known but is also involved in septic disseminated intravascular coagulation (DIC). Here we investigated the ADAMTS13 activity, VWF and VWF propeptide (VWFpp) antigens in 69 patients with DIC, 143 with non-DIC, 21 with thrombotic thrombocytopenic purpura (TTP) and 23 with atypical hemolytic uremic syndrome (aHUS) for diagnosis of DIC.The plasma ADAMTS13 activity was significantly low in patients with DIC, and the plasma levels of VWF and VWFpp antigens, were the highest in these patients, but there were no significant differences in the plasma VWFpp levels between the patients with DIC and those with aHUS. The difference in the plasma ADAMTS13 activity, the VWF and VWFpp antigens between DIC and non-DIC cases was significant in those with infectious and malignant diseases, but the difference in the VWFpp/ VWF ratio were significant only in subjects with infectious diseases. As an indicator for prognosis, the plasma levels of VWFpp were significantly higher in non-survivors than in survivors. Then, VWFpp/ VWF ratio and VWFpp/ADAMATS13 ratio will be potent informative indicators in DIC.These findings suggest that ADAMTS13/VWF profiles may have important roles in the pathogenesis of DIC, and that ADAMTS13 and VWFpp are useful indicators for the diagnosis and prognosis of DIC.     

Expression of the drug-dependent antigen for quinine-dependent antiplatelet antibodies on GP IIIa but not that on GPIb, IIb or IX on human endothelial cells

In quinine- and quinidine-induced thrombocytopenic purpura IgG antibodies are known to react in a drug-dependent manner with different combinations of surface glycoproteins (GP) Ib, IIb, IIIa and IX. Because endothelial cells share a number of properties of platelets, including the presence of GP IIIa and GPIb-like proteins, we have compared these two cell types for their quinine-dependent IgG binding abilities. By immunoblotting of endothelial cells, quinine-dependent IgG binding from four patient sera was observed only to a 93 kDa component corresponding to GP IIIa. Strong IgG binding independent of the drug was found at 170-180 kDa. Thus endothelial cells express the GP IIIa quinine-dependent epitope on platelet GP IIIa, but not those on other platelet glycoproteins.     

Thrombotic microangiopathy]

Thrombotic microangiopathies (TMAs) are characterized by thrombocytopenia, microangiopathic hemolytic anemia, and organ failure (mostly renal dysfunction). TMA includes thrombotic thrombocytopenic purpura (TTP) with predominant neurological signs and hemolytic uremic syndrome (HUS) with predominant renal dysfunction, but they are often indistinguishable each other with the clinical signs alone. Recent availability of von Willebrand factor-cleaving protease or ADAMTS13 activity has defined that TTP is a syndrome frequently associated with a deficient ADAMTS13 activity with or without its inhibitors (autoantibodies), whereas HUS has almost the normal activity. Here, we present two cases of TMA, who were initially diagnosed as "multiple sclerosis" because of the fluctuated neurological signs. Case 1 was a 54-year-old male and case 2 was a 30-year-old female. During their clinical course, they accompanied thrombocytopenia, to which the etiology left undetermined in case 1, but case 2 was suspected DIC because she had such past history. Prophylactic infusion of platelet concentrates to both cases dramatically aggravated their clinical signs. Case 1 was diagnosed to be intravascular lymphoma complicated with acquired TTP, after showing a deficient ADAMTS13 activity. Case 2 was unable to assay ADAMTS13 activity, but later the autopsy revealed the presence of multiple hyaline membrane thrombosis in many organs, together with a lack of demyelinating lesions, solely confirming a diagnosis of TMA.     

An update on the pathogenesis and management of acquired thrombotic thrombocytopenic purpura

PURPOSE OF REVIEW: Thrombotic thrombocytopenic purpura, a clinical syndrome characterized by thrombocytopenia and microangiopathic haemolytic anaemia, was almost universally fatal until the introduction of plasma exchange therapy in the 1970s. Current outcomes have improved dramatically with the initiation of prompt plasma exchange, a treatment routinely used without any real understanding of why it is effective. RECENT FINDINGS: Recent advances suggest that a deficiency of a specific plasma metalloprotease, responsible for the physiological processing of von Willebrand factor multimers, plays a substantial role in the pathogenesis of congenital and acquired idiopathic thrombotic thrombocytopenic purpura. The von Willebrand factor-cleaving protease has now been identified as a new member of the ADAMTS family of metalloproteases, designated ADAMTS13. The acquired form of thrombotic thrombocytopenic purpura is associated with inhibitory autoantibodies against ADAMTS13, and the congenital chronic relapsing form is caused by mutations in the ADAMTS13 gene, resulting in a constitutional deficiency. Plasma exchange has been proved to be the most important therapy in thrombotic thrombocytopenic purpura, but clinical data for adjunctive therapies, such as corticosteroids, antiplatelet drugs and other immunosuppressive agents often used in combination with plasma exchange, are less well defined. SUMMARY: Recent advances in our understanding of the pathological mechanisms of thrombotic thrombocytopenic purpura not only provide a rationale for the previously empirical plasma exchange therapy (removal of the inhibitory antibodies and replacement of the deficient protease from the plasma infused), but may also help in developing more rational and targeted treatment strategies. This review discusses the clinical presentation, pathophysiology and current management of thrombotic thrombocytopenic purpura.     

Inherited ADAMTS13 deficiency (Upshaw-Schulman syndrome): A short review

Congenital thrombotic thrombocytopenic purpura (TTP), also known as Upshaw-Schulman syndrome, is associated with an inherited deficiency of ADAMTS13, a von Willebrand factor-cleaving protease. It is a rare, life-threatening disorder characterized by thrombocytopenia, hemolytic anemia, neurological symptoms, renal dysfunction, and fever resulting from formation of platelet thrombi within the microvasculature. Patients have initial episodes mainly during infancy or early childhood, and are conventionally treated with fresh frozen plasma. However, a more appropriate approach based on recombinant ADAMTS13 is slated to begin shortly. Mutations throughout the ADAMTS13 have been identified in congenital TTP patients. The prevalence of this entity is probably underestimated because it is often not suspected, the clinical course is usually heterogeneous and most of the symptoms are common to other diseases. The present review summarizes our current knowledge about Upshaw-Schulman syndrome.     

Platelet activation rather than endothelial injury identifies risk of thrombosis in subjects positive for antiphospholipid antibodies

BACKGROUND: Anti-phospholipid antibodies (APLA) are often associated with thrombosis, defining the antiphospholipid syndrome (APS) but it remains unclear why many subjects who are positive for APLA chiefly anti-cardiolipin (aCL) or anti-beta2GPI (abeta2GPI) do not develop thrombosis. A related question addressed in this study is whether the target of cellular injury in APS is predominately platelets or endothelial cells (EC). METHODS: aCL and abeta2GPI were determined by ELISA in 88 patients, 60 of whom were thrombotic and 28 non-thrombotic. Platelet activation was measured by CD62P and by concentration of platelet microparticles (PMP) and EC activation was assessed by endothelial microparticles (EMP), both by flow cytometry. Lupus anticoagulant (LAC) was measured in the hospital laboratory. RESULTS: There was no difference in frequency of aCL or abeta2GPI, neither IgG or IgM, between the thrombotic and non-thrombotic groups. Both groups showed elevated EMP compared to controls but this did not differ between thrombotic and non-thrombotic groups. In contrast, PMP were not significantly elevated in non-thrombotic but were elevated in thrombotic compared to non-thrombotic (p=0.03) and controls. CD62P, an independent marker of platelet activation, was also elevated in thrombotic vs. non-thrombotic. There was a trend for increased LAC in the thrombotic group but not significant. CONCLUSION: Although all subjects had evidence of endothelial activation, only platelet activation differed between thrombotic and non-thrombotic. This supports the hypothesis that platelet activation predisposes to thrombosis in the presence of chronic EC activation. These data also raise the possibility of distinguishing risk-prone APLA-positive individuals.     

Platelet-derived microparticles during and after acute coronary syndrome

As microparticles are shedded upon platelet activation, and may be used to assess platelet function, we measured plasma concentrations of platelet-derived microparticles (PMPs) during and after an acute coronary syndrome (ACS). Fifty-one patients with ACS were investigated at admission, within 24 hours (before coronary angiography), and six months later. Sixty-one sex- and age-matched healthy controls were investigated once. PMPs were defined as particles <1.0 μm in size, negative to phalloidin (labels cell-fragments), and positive to CD61. Exposure of phosphatidylserine (PS+), CD62P and CD142 were also measured. Plasma concentrations of PS+PMPs exposing CD61, CD62P and CD142 were elevated 2.5, 6.0-, and 5.0-fold at admission (p<0.001 for all, compared to controls; aspirin only), decreased significantly 24 hours later following initiation of treatment with clopidogrel and subcutaneous anticoagulation (p<0.001 for all), and decreased even further six months later (p<0.01 for all). However, PS+PMPs exposing CD62P or CD142 were still between 1.2-and 2.3-fold higher than in controls (p<0.001 for both). The pattern for PS-PMPs during and after the ACS was very similar to that for PS+PMPs although the numbers were approximately 1/3 lower. In conclusion, PMP concentrations follow the pattern of platelet activation during and after an ACS. Decreased concentrations are observed after initiation of antithrombotic treatment, but PMP exposing CD62P or CD142 are still elevated after six months. Flow cytometric measurements of PMP in frozen-thawed samples enable studies of platelet function in larger clinical trials.     

Assessment of in vitro-generated platelet microparticles using a modified flow cytometric strategy.

Quantification of platelet microparticles (PMPs) may be a useful marker for the detection of in vivo platelet activation. Optimisation of flow cytometric methods for detection and quantification of PMPs has not been systemically evaluated. This study reports the optimisation of flow cytometric procedures for the detection of PMPs, the determination of limits of size detection using microbeads, and the characterisation of PMP generation by in vitro activation of platelets using collagen and adenosine 5' diphosphate (ADP). Fluorescent and plain microbeads proved useful for defining the limits of the flow cytometer in detecting PMPs. A systematic calibration of the forward scatter (FS) threshold parameter (size) of the flow cytometer using microbeads allowed for the detection of very small particles (down to 0.1 microm diameter). PMPs generated in vitro using ADP and collagen were reliably detected by flow cytometry using monoclonal antibodies (MAb) directed towards platelet surface membrane glycoproteins (Gp). The PMP events were detected in the FS low (i.e., small size events) and fluorescence (FL) high (i.e., platelet Gp MAb-labelled events) region. PMPs of different size profiles were observed for each of the agonists. Flow cytometry can be used as a tool in the assessment of PMPs. As detection of particles of this type is at the limit of resolution of flow cytometers, careful attention is required with the choice of platelet-specific MAb, isotype control, and optimisation of procedure setup and performance.     

Therapeutic plasma exchange and plasma infusion in thrombotic microvascular syndromes

Plasma exchange (PE) is the most important treatment in thrombotic microangiopathies (TMAs) mainly encompassing thrombotic thrombocytopenic purpura (TTP) and adult hemolytic syndrome (HUS). This therapeutic measure has substantially improved clinical outcome. One plasma volume corresponding to 40 ml/kg of body weight is exchanged daily until the platelet count is above 150 x 10(9)/l or 100 x 10(9)/l and continues to rise or remains constantly after cessation of treatment. Exacerbations and late recurrences demand reapplication of daily PE. Twice daily PEs are initiated if the response to initial treatment is poor. The importance of additional or alternate measures including glucocorticoids, antiplatelet agents, splenectomy, intravenous immunoglobulins, protein A columns, vincristine, cyclosporine, and cyclophosphamide is uncertain. Whether cryosupernatant plasma (CSP) or solvent/detergent-treated (SDP) plasma is superior to standard fresh frozen plasma (FFP) remains to be determined. Methylene blue-treated plasma (MBP) seems to be less effective than standard FFP.     

Ocular manifestations of thrombotic thrombocytopenic purpura

We report a case of thrombotic thrombocytopenic purpura (TTP) in a 47-year-old woman, who presented fluctuating visual disturbances which had developed over the last six months. An antiphospholipid syndrome was suspected and intravenous heparin treatment was started. One week later, hemolytic anemia and renal insufficiency occurred. Severe deficiency of von Willebrand factor-cleaving protease was found and a diagnostic of TTP was made. The clinical outcome was favorable after treatment with plasmapheresis and fresh frozen plasma. Diagnosis, etiology and treatment of this life-threatening disease are discussed.     

The potential role of platelet microparticles in atherosclerosis

The release of microvesicles ('platelet microparticles', PMPs) by activated platelets has been shown to be an integral part of the thrombotic process. PMPs are believed to mediate many biological processes as they possess various platelet membrane proteins and bioactive lipids. Of note, there is a growing body of evidence that PMPs are involved in all stages in the pathobiology of atherosclerosis. In addition to their role in thrombosis, PMPs may also have a pro-inflammatory effect, which promotes the development of atherosclerosis. Also, bioactive lipids in PMPs have been shown to have important effects on angiogenesis.This review summarises the various studies on the possible role of PMPs in the progression of atherosclerosis.