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The primary B-cell epitopes of factor VIII (fVIII) are in the A2 and C2 domains. Within the C2 domain, antibody epitope and kinetics are more important than inhibitor titer in predicting pathogenicity in a murine bleeding model. To investigate this within the A2 domain, the pathogenicity of a diverse panel of antihuman fVIII A2 domain monoclonal antibodies (MAbs) was tested in the murine model. MAbs were injected into hemophilia A mice, followed by injection of human B domain-deleted fVIII. Blood loss after a 4-mm tail snip was measured. The following anti-A2 MAbs were tested: high-titer type 1 inhibitors 4A4, 2-76, and 1D4; 2-54, a high-titer type 2 inhibitor; B94, a type 2 inhibitor; and noninhibitory MAbs GMA-012, 4C7, and B25. All high-titer type 1 MAbs produced blood loss that was significantly greater than control mice, whereas all non-inhibitory MAbs produced blood loss that was similar to control. The type 2 MAbs were not pathogenic despite 2-54 having an inhibitor titer of 34 000 BU/mg immunoglobulin G. In addition, a patient with a high-titer type 2 anti-A2 inhibitor who is responsive to fVIII is reported. The discrepancy between inhibitor titer and bleeding phenotype combined with similar findings in the C2 domain stress the importance of inhibitor properties not detected in the standard Bethesda assay in predicting response to fVIII therapy., (© 2016 by The American Society of Hematology.)

Thrombocytopenia is a common finding in sepsis and associated with a worse outcome. We used a mouse model of pneumonia-derived sepsis caused by the human pathogen Klebsiella pneumoniae to study the role of platelets in host response to sepsis. Platelet counts (PCs) were reduced to less than a median of 5 × 10(9)/L or to 5 to 13 × 10(9)/L by administration of a depleting antibody in mice infected with Klebsiella via the airways. Thrombocytopenia was associated with strongly impaired survival during pneumonia-derived sepsis proportional to the extent of platelet depletion. Thrombocytopenic mice demonstrated PC-dependent enhanced bacterial growth in lungs, blood, and distant organs. Severe thrombocytopenia resulted in hemorrhage at the primary site of infection, but not in distant organs. PCs of 5 to 13 × 10(9)/L were sufficient to largely maintain hemostasis in infected lungs. Thrombocytopenia did not influence lung inflammation or neutrophil recruitment and did not attenuate local or systemic activation of coagulation or the vascular endothelium. PCs <5 × 10(9)/L even resulted in enhanced coagulation and endothelial cell activation, which coincided with increased proinflammatory cytokine levels. In accordance, low PCs in whole blood enhanced Klebsiella-induced cytokine release in vitro. These data suggest that platelets play an important role in host defense to Klebsiella pneumosepsis., (© 2014 by The American Society of Hematology.)

In this issue of Blood, de Stoppelaar et al further unravel the relevance of platelets in a mouse model of pneumonia-derived sepsis by illustrating how platelets dynamically modulate infection and the inflammatory response.

Long-term follow-up of children with immune thrombocytopenia (ITP) indicates that the majority undergo remission and severe thrombocytopenia is infrequent. Details regarding bleeding manifestations, however, remain poorly categorized. We report here long-term data from the Intercontinental Cooperative ITP Study Group Registry II focusing on natural history, bleeding manifestations, and management. Data on 1345 subjects were collected at diagnosis and at 28 days, 6, 12, and 24 months thereafter. Median platelet counts were 214 × 10(9)/L (interquartile range [IQR] 227, range 1-748), 211 × 10(9)/L (IQR 192, range 1-594), and 215 × 10(9)/L (IQR 198, range 1-598) at 6, 12, and 24 months, respectively, and a platelet count <20 × 10(9)/L was uncommon (7%, 7%, and 4%, respectively). Remission occurred in 37% of patients between 28 days and 6 months, 16% between 6 and 12 months, and 24% between 12 and 24 months. There were no reports of intracranial hemorrhage, and the most common site of bleeding was skin. In patients with severe thrombocytopenia we observed a trend toward more drug treatment with increasing number of bleeding sites. Our data support that ITP is a benign condition for most affected children and that major hemorrhage, even with prolonged severe thrombocytopenia, is rare.

Acquired factor XIII (FXIII) deficiency due to autoantibody against FXIII is a very rare severe hemorrhagic diathesis. Antibodies directed against the A subunit of FXIII, which interfere with different functions of FXIII, have been described. Here, for the first time, we report an autoantibody against the B subunit of FXIII (FXIII-B) that caused life-threatening bleeding in a patient with systemic lupus erythematosus. FXIII activity, FXIII-A(2)B(2) complex, and individual FXIII subunits were undetectable in the plasma, whereas platelet FXIII activity and antigen were normal. Neither FXIII activation nor its activity was inhibited by the antibody, which bound to structural epitope(s) on both free and complexed FXIII-B. The autoantibody highly accelerated the elimination of FXIII from the circulation. FXIII supplementation combined with immunosuppressive therapy, plasmapheresis, immunoglobulin, and anti-CD20 treatment resulted in the patient's recovery. FXIII levels returned to around 20% at discharge and after gradual increase the levels stabilized above 50%.

Acquired hemophilia (AH) is an extremely rare condition in which autoantibodies (inhibitors) against clotting factor VIII induce acute and life-threatening hemorrhagic diathesis because of abnormal blood clotting. The mortality rate of AH is as high as 16%, and current treatment options are associated with adverse side effects. We investigated a therapeutic approach for AH called the modified Bonn-Malmo Protocol (MBMP). The aims of MBMP include suppression of bleeding, permanent elimination of inhibitors, and development of immune tolerance, thereby avoiding long-term reliance on coagulation products. The protocol included immunoadsorption for inhibitor elimination, factor VIII substitution, intravenous immunoglobulin, and immunosuppression. Thirty-five high-titer patients with critical bleeding who underwent MBMP were evaluated. Bleeding was rapidly controlled during 1 or 2 apheresis sessions, and no subsequent bleeding episodes occurred. Inhibitor levels decreased to undetectable levels within a median of 3 days (95% confidence interval [95% CI], 2-4 days), factor substitution was stopped within a median of 12 days (95% CI, 11-17 days), and treatment was completed within a median of 14 days (95% CI, 12-17 days). Long-term follow-up (7 months-7 years) showed an overall response rate of 88% for complete remission (CR). When cancer patients were excluded, the CR rate was 97%.

The authors examined the efficacy of 2-chlorodeoxyadenosine (2-CDA) in the treatment of refractory inhibitors to factor VIII in persons without hemophilia. The drug was administered to 6 patients at a dose of 0.1 mg/kg as a 24-hour continuous infusion for a total of 7 days each cycle. An average of 3 immunosuppressive regimens per patient had been administered prior to enrollment in this study. The median inhibitor titer against human and porcine factor VIII before treatment with 2-CDA was 31 Bethesda units (BUs) and 9 BU, respectively. The median inhibitor titer against human and porcine factor VIII after treatment was 3.5 BU and 1.5 BU, respectively. The median time to reach nadir inhibitor titer in this study was 137 days, whereas the median time to reach a 50% increase in factor VIII was 117 days. No major toxicity was observed in any patient in this study. Patients with acquired inhibitors to factor VIII refractory to conventional immunosuppressive therapy may respond favorably to 2-CDA.

Hirudin, a potent and specific thrombin inhibitor, is a protein of nonhuman origin and therefore potentially immunogenic. The primary objectives of this investigation were to determine the incidence of antihirudin antibodies (ahir-ab) in patients with heparin-induced thrombocytopenia (HIT) who received lepirudin as parenteral anticoagulation and to determine the incidence of death, limb amputation, new thromboembolic complications (TECs), and major hemorrhage in patients who had ahir-ab, compared with patients who were ahir-ab negative. The investigation used data from 2 prospective multicenter studies with the same study protocol, in which HIT patients received 1 of 4 intravenous lepirudin dosage regimens. The treatment duration was 2 to 10 days. Ahir-ab were determined by a newly developed enzyme-linked immunosorbent assay (ELISA). Eighty-seven of 196 evaluable patients (44.4%) had ahir-ab of the IgG class. Development of ahir-ab was dependent on the duration of treatment (ahir-ab-positive patients 18.6 days vs ahir-ab-negative patients 11.8 days; P =.0001). Fewer ahir-ab-positive than ahir-ab-negative patients died (P =.001). Ahir-ab did not cause an increase in limb amputation (P =.765), new TECs (P >.99), or major bleedings (P =.549). In 23 of 51 (45.1%) evaluable patients in whom ahir-ab developed during treatment with lepirudin ( = 12% of all lepirudin treated patients), the ahir-ab enhanced the anticoagulatory effect of lepirudin. Ahir-ab are frequent in patients treated with lepirudin for more than 5 days. Ahir-ab are the first example for a drug-induced immune response causing enhanced activity of a drug. Therefore, during prolonged treatment with lepirudin, anticoagulatory activity should be monitored daily to avoid bleeding complications.

The pathogenic effects of antiplatelet antibodies were investigated in mice. Monoclonal antibodies (mAbs) of different immunoglobulin G subclass directed against mouse GPIIbIIIa, GPIIIa, GPIbalpha, GPIb-IX, GPV, and CD31 were generated and characterized biochemically. MAbs against GPIb-IX, GPV, CD31, and linear epitopes on GPIIIa had mild and transient effects on platelet counts and induced no spontaneous bleeding. Anti-GPIbalpha mAbs induced profound irreversible thrombocytopenia (< 3% of normal) by Fc-independent mechanisms but only had minor effects on hematocrits. In contrast, injection of intact mAbs, but not F(ab)(2) fragments, against conformational epitopes on GPIIbIIIa, induced irreversible thrombocytopenia, acute systemic reactions, hypothermia, decreased hematocrits, and a paradoxical loss of surface GPIIbIIIa on platelets in vivo, the latter suggesting the formation of platelet-derived microparticles. Blockage of platelet-activating factor receptors inhibited the acute reactions, but not thrombocytopenia, loss of GPIIbIIIa, and decreases in hematocrits. Repeated injections of low doses of anti-GPIIbIIIa antibodies resulted in profound thrombocytopenia and bleeding, whereas no acute systemic reactions were observed. These data strongly suggest that the identity of the target antigen recognized by antiplatelet antibodies determines the mechanisms of platelet destruction and the severity of bleeding in mice, the latter depending on previously unrecognized anti-GPIIbIIIa-specific inflammatory mechanisms.