Prolonged PFA-100 Closure Times Research Articles

Identification of two novel mutations in RASGRP2 affecting platelet CalDAG-GEFI expression and function in patients with bleeding diathesis

The RASGRP2 gene encodes the Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbβ3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn2+-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.

Comparison of coated-platelet levels in patients with essential thrombocythemia with and without hydroxyurea treatment

Essential thrombocythemia (ET) is an acquired myeloproliferative disorder with sustained increase of platelet count. This disease may be associated with thrombotic or bleeding complications due to the altered number and function of platelets. Coated-platelets produced by a simultaneous activation of collagen and thrombin represent a subpopulation of activated platelets with high prothombinase activity and the retention of several α-granule-derived coagulation factors on their surface. There is a growing body of evidence for a relationship between variable levels of coated-platelets and different hemostatic alterations. However, no data are available on coated-platelet formation in the pathogenesis of ET in the presence or absence of treatment. The levels of coated-platelets in 43 ET patients (15 non-treated and 28 hydroxyurea-treated) without known thrombotic or hemorrhagic complications were analyzed using flow cytometry. These results were compared with data of 31 healthy individuals. In addition, platelet function was analyzed with PFA-100 analysis, and P-selectin (CD62) positivity was also measured by flow cytometry. Increased P-selectin expression was detected with prolonged PFA-100 closure times in the ET group; however, significantly lower levels of coated-platelets were found in non-treated ET patients compared to controls (23.1 ± 8.8% vs. 37.6 ± 12.7%, p = 0.0008). This tendency was more evident in patients with JAK2-V617F mutation. Patients on hydroxyurea treatment had elevated coated-platelet levels (34.1 ± 12.3%) close to the normal value. In conclusion, lower than normal levels of coated-platelets were generated in ET, which were significantly (p = 0.0008) increased by hydroxyurea treatment. We suppose that abnormal coated-platelet level may also contribute to platelet dysfunction in ET.

Effects of carprofen, meloxicam and deracoxib on platelet function in dogs

ObjectiveTo determine effects of anti-inflammatory doses of COX-2 selective NSAIDs carprofen, meloxicam, and deracoxib on platelet function in dogs and urine 11-dehydro-thromboxane B2. Study designRandomized, blocked, crossover design with a 14-day washout period. AnimalsHealthy intact female Walker Hounds aged 1–6 years and weighing 20.5–24.2 kg. MethodsDogs were given NSAIDs for 7 days at recommended doses: carprofen (2.2 mg kg−1, PO, every 12 hours), carprofen (4.4 mg kg−1, PO, every 24 hours), meloxicam (0.2 mg kg−1, PO, on the 1st day then 0.1 mg kg−1, PO, every 24 hours), and deracoxib (2 mg kg−1, PO, every 24 hours). Collagen/epinephrine and collagen/ADP PFA-100 cartridges were used to evaluate platelet function before and during and every other day after administration of each drug. Urine 11-dehydro-thromboxane B2 was also measured before and during administration of each drug. ResultsAll NSAIDs significantly prolonged PFA-100 closure times when measured with collagen/epinephrine cartridges, but not with collagen/ADP cartridges. The average duration from drug cessation until return of closure times (collagen/epinephrine cartridges) to baseline values was 11.6, 10.6, 11 and 10.6 days for carprofen (2.2 mg kg−1 every 12 hours), carprofen (4.4 mg kg−1 every 24 hours), meloxicam and deracoxib, respectively. Conclusions and clinical relevanceOral administration of some COX-2 selective NSAIDs causes detectable alterations in platelet function in dogs. As in humans, PFA-100 collagen/ADP cartridges do not reliably detect COX-mediated platelet dysfunction in dogs. Individual assessment of platelet function is advised when administering these drugs prior to surgery, particularly in the presence of other risk factors for bleeding.

The effects of cytochalasin D and abciximab on hemostasis in canine whole blood assessed by thromboelastography and the PFA-100® platelet function analyzer system

The selective inhibition of platelet function in whole blood coagulation testing may allow insights into the nature of hypercoagulability in dogs with critical illness. To determine the effects of cytochalasin D and abciximab on hemostatic parameters in canine citrated whole blood, an in-vitro study was designed using thromboelastography (TEG) and a platelet function analyzer (PFA-100®). 8 clinically healthy mixed breed dogs donated blood that was anticoagulated with 3.2% sodium citrate in a 9:1 blood-to-citrate ratio. Addition of cytochalasin D to citrated whole blood from 6 dogs at concentrations ranging from 0 µg/ml to 10 µg/ml caused a maximal reduction of TEG maximum amplitude (MA) at a concentration of 7.5 µg/ml (52.7 ± 4.3 to 14.3 ± 7.8 mm). Addition of abciximab to canine citrated whole blood at concentrations of either 20 µg/ml or 40 µg/ml did not affect the TEG tracing; however, addition of abciximab to citrated canine whole blood at concentrations of 10 µg/ml and 20 µg/ml significantly prolonged PFA-100 closure times (72.5 ± 15 to 149.2 ± 91 sec and 275.6 ± 54 sec, respectively, P < 0.04). Inhibition of canine platelet function by cytochalasin D is demonstrated by TEG, but abciximab did not change TEG tracings. Abciximab does, however, inhibit platelet aggregation under shear stress as measured by the PFA-100. Inhibition of canine platelet function with cytochalasin D may allow further TEG studies in dogs with clinical disease.

Severe Bleeding Complications during Antiepileptic Treatment with Valproic Acid in Children: A Description of Four Cases.

Sodium valproat (VPA) is a commonly used antiepileptic drug in children. It is known to cause a variety of different haematological side effects, including thrombocytopenia, a decrease in plasma fibrinogen, von Willebrand factor (VWF)- and factor XIII, impaired platelet aggregation with ADP and Collagen and prolonged PFA-100 closure times. The mechanisms responsible for such a variety of haematological abnormalities are still poorly understood and controversy exists about their clinical relevance and the risk for significant bleeding. We report on 4 children with severe bleeding complications during VPA treatment that had been followed at our institution.A first child with a cerebral malformation and no history of bleeding developed a subdural haematoma during VPA treatment without trauma. Additional studies revealed decreased von Willebrand factor levels that persisted with episodes of severe epistaxis also after VPA was stopped. A second infant suffered from herpes encephalitis with generalised seizures and died from an intracranial bleeding a day after a VPA loading dose was administred. Laboratory studies revealed prolonged PFA100 closure times. A third child with a therapeutic VPA serum-levels developed asymptomatic mild thrombocytopenia (around 100 G/l); unexpectedly in this child a tonsillectomy was complicated with sever diffuse haemorrhage that required red cell transfusion and intensive care treatment. Finally, a forth child who had been on VPA therapy for years causing prolonged PFA100 closure times, developed a large spinal epidural haematoma during epidural anaesthesia three days after urological surgery. This child had received VWF containing factor concentrate preoperatively only. For the bleeding additional VWF containing factor concentrate was given, the catheter was removed and the child recovered completely.Paediatricians, neurologists and anesthesists should be aware of the haemostatic side effects of VPA that can lead to significant bleeding. Before starting VPA therapy, patients should be evaluated for the presence of a congenital bleeding disorder. Preoperatively for any surgical intervention and in case of the occurrence of bleeding symptoms the patients have to be further evaluated for a VPA-induced disorder of the plasmatic haemostasis or an impaired platelet function. Haemostatic side effects of VPA can occur at therapeutic VPA serum levels and are found at any time during the course of VPA treatment. VPA has to be used cautiously in severely ill children and in intensive care patients and the risk of bleeding of any surgical or anesthesiological procedure has to be evaluated. Treatment options for VPA induced haemostatic abnormalities include plasma derived factor concentrates that contain VWF and the use of antifibrinolytic agents. Further studies have to assess the feasibility of standard treatment with the vasopressin analog DDAVP for this group of patients taking into account their high risk for seizures.

The role of CD40 in CD40L- and antibody-mediated platelet activation

Our initial finding that CD40- and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhsCD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhsCD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28-5 showed less potential in blocking rhsCD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhsCD40L, G28-5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcgammaRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhsCD40L induced strong Fcgamma RII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.

The CD40/CD40 Ligand (CD40L) Pathway Plays a Role in Primary Hemostasis and Platelet Function.

Previous studies have suggested a role for platelet CD40L in thrombosis and atherosclerosis. However, there are contradictory reports on the biologic activity of its soluble variant (sCD40L) and the involved receptor signaling pathways (CD40 vs αIIbβ3). Furthermore, CD40L mAb-associated thromboembolic complications in recent human and animal studies have raised additional questions about the pro-thrombotic properties of this molecule. This study was conducted to further investigate the function of the CD40/CD40L dyad in primary hemostasis and platelet function. CD40−/− and CD40L−/− mice and mice deficient for both genes (“double knock-out”) showed prolonged tail vein bleeding and platelet function analyzer (PFA-100) closure times as compared to their wild-type littermates, indicating an inherited defect in platelet function. Recombinant human sCD40L (rsCD40L), chemical cross-linking of which yielded a single reaction product compatible with a trimeric structure of the protein in solution, bound to CD40 on resting platelets and induced CD62P (P-selectin) expression in a concentration-dependent manner (0–5 μg/ml) from 1±1 to 23±5% positivity (means±SD, n=4–8; P<0.01). This response was completely abolished by CD40 mAb M3 and not affected by blocking the β3 integrin (CD61) with abciximab. In contrast, CD40 mAb G28-5 significantly enhanced rsCD40L-induced CD62P expression to 51±5%. This agonistic effect was strongest when G28-5 was added to the platelets after rsCD40L. Pre-incubation of rsCD40L with CD40L mAb M90 also had a potentiating effect, showing an inverted V-shaped dose response curve with maximum levels of CD62P+ platelets (60–95%) at a molar M90/rsCD40L ratio of 1/3. G28-5 and M90 alone had no effect, but their combination in the presence of rsCD40L proved synergistic. Experiments with corresponding F(ab') fragments and the FcγRII-inhibitory mAb IV.3 demonstrated that G28-5- and M90-mediated additional platelet activation resulted from signaling through FcγRII cross-linked to CD40 and rsCD40L bound to CD40 on the platelet surface, respectively. The CD40L mAb TRAP-1 showed similar characteristics to M90, but its synergistic effect with rsCD40L was less pronounced. Platelet activation by rsCD40L in combination with G28-5 and/or M90 also induced morphological shape changes, fibrinogen binding, dense granule release, microparticle generation, and monocyte-platelet-conjugate formation. Interestingly, consistent with their platelet function-modulating effects, M3 but not G28-5 prolonged PFA-100 closure times of normal human blood. This work provides genetic evidence for a role of CD40 and CD40L in primary hemostasis. Our mechanistic studies further suggest that CD40-mediated platelet activation by CD40L, in its membrane and/or soluble form, may be at least partially involved in this process. The results also offer a potential explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in patients with systemic lupus erythematosus, an inflammatory autoimmune disease characterized by elevated levels of circulating sCD40L. The underlying pathomechanism, in which sCD40L as a platelet-derived, homotrimeric protein causes multimeric clustering of bivalent antibodies on the platelet surface with subsequent FcγRII-mediated platelet activation, resembles other known immune thrombophilias such as the heparin-induced thrombocytopenia (HIT) syndrome.

Von Willebrand Disease in a Pediatric-based Population - Comparison of Type 1 Diagnostic Criteria and Use of the PFA-100® and a von Willebrand Factor/Collagen-binding Assay

Definitive diagnosis of type 1 von Willebrand Disease (VWD) remains a problem. Provisional consensus guidelines for the diagnosis of definite and possible type 1 VWD were prepared by the Scientific Subcommittee on von Willebrand factor (VWF) of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) during the 1996 annual meeting for the specific purpose of further evaluation in retrospective and prospective studies by a Working Party on Diagnostic Criteria (1996 Annual Report of the SSC/ISTH Subcommittee on VWF). In the first phase of this study, we compared 2 definitions of type 1 VWD. each with 3 criteria: significant bleeding history, laboratory investigations, and family history. Using the ISTH consensus guidelines for type 1 VWD definition, significantly fewer patients were diagnosed with definite type 1 disease as compared to our "in house" Hospital for Sick Children (HSC) criteria (4 vs. 31). While we recognize that the provisional ISTH consensus guidelines were not intended for clinical use, we believe that the results of our studies are of interest and will assist in any future refinements to the ISTH guidelines. In the second phase of this study, we investigated the utility of 2 new tests, a laboratory screening test and a functional test, for VWD in our well characterized, pediatric-based population. The Platelet Function Analyzer (PFA-100) provides an in vitro measure of primary hemostasis under conditions of high shear, using disposable cartridges containing collagen and either epinephrine or ADP. All tested subjects with types 2 or 3 VWD had prolonged PFA-100 closure times (CTs) with both cartridge types (n = 17) and prolonged bleeding times (n = 14). In subjects with definite type 1 VWD, 20/24 (83%) had prolonged CTs with the collagen/ADP cartridge (19/24 (79%) with collagen/epinephrine), compared with 7/26 (27%) with prolonged bleeding times. In subjects with definite types 1, 2, or 3 VWD, collagen/ADP CTs were abnormal in 37/41 subjects, giving an overall sensitivity of 90%. With this high sensitivity, the PFA-100 is a better screening test for VWD than the bleeding time. We also tested a VWF collagen-binding assay (VWF:CBA) as a functional test for VWF, in comparison with the more routinely-used ristocetin cofactor assay (VWF:RC0). The VWF:CBA is based on an ELISA technique, which has the potential to be more reproducible than the VWF:RC0. We found that the VWF:CBA detected 43/49 (88%) subjects with definite types 1, 2, or 3 VWD, performing as well as the VWF:RC0, that detected 42/48 (88%). We also showed that, used in conjunction with VWF antigen levels, the VWF:CBA may be useful in classification of VWD subtypes.