Sarah E. Sartain

Thrombotic Microangiopathies and the Linkage between von Willebrand Factor and the Alternative Complement Pathway

Molecular linkages between von Willebrand factor (VWF) and the alternative complement pathway (AP) have recently been discovered. Endothelial cell (EC)-anchored ultra-large (UL) VWF multimeric strings function as an activating surface for the AP. C3 (in active C3b form) binds to the EC-anchored ULVWF strings, and promotes the assembly of C3bBb (C3 convertase) and C3bBbC3b (C5 convertase). These linkages may help to explain enigmatic clinical problems related to thrombotic microangiopathies, including some cases of refractory thrombotic thrombocytopenic purpura (TTP), TTP associated with only mild-modest deficiencies of ADAMTS-13, the provocation (or exacerbation) of acute episodes in patients with the atypical hemolytic uremic syndrome, and thrombosis in paroxysmal nocturnal hemoglobinuria. Recent experiments have also demonstrated that complement factor H performs a dual role: participating in regulation of the AP by binding to EC-anchored ULVWF strings; and functioning as a reductase to decrease the size of soluble VWF multimers.

Ultralarge von Willebrand factor-induced platelet clumping and activation of the alternative complement pathway in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndromes.

The molecular linkage between ultralarge (UL) von Willebrand factor (VWF) multimers and the alternative complement pathway (AP) has recently been described. Endothelial cell (EC)-secreted and anchored ULVWF multimers (in long stringlike structures) function as both hyperadhesive sites that initiate platelet adhesion and aggregation and activating surfaces for the AP. In vitro, the active form of C3, C3b binds to the EC-anchored ULVWF multimeric strings and initiates the assembly on the strings of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b). In vivo, activation of the AP via this mechanism proceeds all the way to generation of terminal complement complexes (C5b-9).

TNF Regulates Essential Alternative Complement Pathway Components and Impairs Activation of Protein C in Human Glomerular Endothelial Cells.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy with severe renal injury secondary to an overactive alternative complement pathway (AP). aHUS episodes are often initiated or recur during inflammation. We investigated gene expression of the surface complement regulatory proteins (CD55, CD59, CD46, and CD141 [thrombomodulin]) and AP components in human glomerular microvascular endothelial cells (GMVECs) and in HUVECs, a frequently used investigational model of endothelial cells. Surface complement regulatory proteins were also quantified by flow cytometry. All experiments were done with and without exposure to IL-1β or TNF. Without cytokine stimulation, we found that GMVECs had greater AP activation than did HUVECs. With TNF stimulation, THBD gene expression and corresponding CD141 surface presence in HUVECs and GMVECs were reduced, and gene expression of complement components C3 (C3) and factor B (CFB) was increased. Consequently, AP activation, measured by Ba production, was increased, and conversion of protein C (PC) to activated PC by CD141-bound thrombin was decreased, in GMVECs and HUVECs exposed to TNF. IL-1β had similar, albeit lesser, effects on HUVEC gene expression, and it only slightly affected GMVEC gene expression. To our knowledge, this is the first detailed study of the expression/display of AP components and surface regulatory proteins in GMVECs with and without cytokine stimulation. In aHUS patients with an underlying overactive AP, additional stimulation of the AP and inhibition of activated PC–mediated anticoagulation in GMVECs by the inflammatory cytokine TNF are likely to provoke episodes of renal failure.

Regulatory components of the alternative complement pathway in endothelial cell cytoplasm, factor H and factor I, are not packaged in Weibel-Palade bodies.

It was recently reported that factor H, a regulatory component of the alternative complement pathway, is stored with von Willebrand factor (VWF) in the Weibel-Palade bodies of endothelial cells. If this were to be the case, it would have therapeutic importance for patients with the atypical hemolytic-uremic syndrome that can be caused either by a heterozygous defect in the factor H gene or by the presence of an autoantibody against factor H. The in vivo Weibel-Palade body secretagogue, des-amino-D-arginine vasopressin (DDAVP), would be expected to increase transiently the circulating factor H levels, in addition to increasing the circulating levels of VWF. We describe experiments demonstrating that factor H is released from endothelial cell cytoplasm without a secondary storage site. These experiments showed that factor H is not stored with VWF in endothelial cell Weibel-Palade bodies, and is not secreted in response in vitro in response to the Weibel-Palade body secretagogue, histamine. Furthermore, the in vivo Weibel-Palade body secretagogue, DDAVP does not increase the circulating factor H levels concomitantly with DDAVP-induced increased VWF. Factor I, a regulatory component of the alternative complement pathway that is functionally related to factor H, is also located in endothelial cell cytoplasm, and is also not present in endothelial cell Weibel-Palade bodies. Our data demonstrate that the factor H and factor I regulatory proteins of the alternative complement pathway are not stored in Weibel-Palade bodies. DDAVP induces the secretion into human plasma of VWF —- but not factor H.

Brain microvascular endothelial cells exhibit lower activation of the alternative complement pathway than glomerular microvascular endothelial cells

Atypical hemolytic uremic syndrome (aHUS) and bone marrow transplantation-associated thrombotic microangiopathy (TA-TMA) are associated with excessive activation of the alternative complement pathway (AP) and with severe renal, but rarely cerebral, microvascular damage. Here, we compared AP activation and regulation in human glomerular and brain microvascular endothelial cells (GMVECs and BMVECs, respectively) unstimulated or stimulated by the proinflammatory cytokine, tumor necrosis factor (TNF). Compared with GMVECs and under both experimental conditions, BMVECs had increased gene expression of the AP-related genes C3, CFB, and C5 and decreased expression of CFD. This was associated with increased expression in BMVECs (relative to GMVECs) of the genes for surface and soluble regulatory molecules (CD46, THBD, CD55, CFI, and CFH) suppressing formation of the AP C3 and C5 convertases. Of note, unlike GMVECs, BMVECs generated extremely low levels of C3a and C5a and displayed decreased activation of the AP (as measured by a lower percentage of Ba generation than GMVECs). Moreover, BMVECs exhibited increased function of CD141, mediating activation of the natural anticoagulant protein C, compared with GMVECs. We also found that the C3a receptor (C3aR) is present on both cell types and that TNF greatly increases C3AR1 expression in GMVECs, but only slightly in BMVECs. Higher AP activation and C3a generation in GMVECs than in BMVECs, coupled with an increase in C3aR production in TNF-stimulated GMVECs, provides a possible explanation for the predominance of renal damage, and the absence of cerebral injury, in individuals with episodes of aHUS and TA-TMA.

Pathophysiology of Thrombotic Thrombocytopenic Purpura

Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized by microangiopathic hemolytic anemia (MAHA), thrombocytopenia, microvascular endothelial injury and thrombosis, as well as end-organ damage particularly of the central nervous system, kidneys, heart, and gastrointestinal tract. The pathogenesis of the disorder is secondary to deficiencies in ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which is responsible for cleaving large multimers of von Willebrand factor (VWF). VWF normally functions to initiate formation of the platelet plug in primary hemostasis. In TTP, absence or deficiency of ADAMTS13 leads to persistence of ultra-large VWF (ULVWF) multimers with subsequent ULVWF-platelet adhesion and development of systemic microvascular thrombosis. Thrombocytopenia is secondary to platelet consumption in the formation of systemic microthrombi; MAHA and schistocytosis develop as a result of mechanical fragmentation as the red blood cells cross these platelet thrombi. Microvascular endothelial injury in TTP is well described, but the pathophysiologic mechanisms for this injury are not well characterized. Proposed mechanisms include direct injury by platelet-VWF thrombi, anti-endothelial cell antibodies, nitric oxide, oxidative stress, and neutrophil activation, as well as activation of the Fas pathway leading to endothelial cell apoptosis. Endothelial injury and thrombosis of the small vessels lead to impaired organ perfusion, further resulting in multi-organ failure, accounting for the significant morbidity and mortality observed in TTP. Severe ADAMTS13 deficiency is caused by congenital ADAMTS13 gene mutations or by acquired production of polyclonal autoantibodies directed against the metalloprotease. Autoantibody development in TTP can be triggered by infections or, more often, occur in the setting of other autoimmune phenomena. Other etiologies of acquired TTP include sepsis, liver disease, pancreatitis, pregnancy, HIV, cancer, organ transplant, and drugs. More recently, both in vitro and in vivo evidence have pointed toward over-activation of the alternative complement pathway, part of the innate immune system, in TTP, suggesting an additional immune mechanism in the pathogenesis of the disorder.

Work efficiency improvement of >90% after implementation of an annual inpatient blood products administration consent form

Paediatric haematology, oncology and bone marrow transplant (BMT) patients frequently require transfusion of blood products. Our institution required a new transfusion consent be obtained every admission. The objectives of this project were to: revise inpatient blood products consent form to be valid for 1 year, decrease provider time spent consenting from 15 to <5 min per admission, and improve provider frustration with the consent process. Over 6 months, we determined the average number of hospitalisations requiring transfusions in a random sampling of haematology/oncology/BMT inpatients. We surveyed nurses and providers regarding frustration levels and contact required regarding consents. Four and 12 months after implementation of the annual consent, providers and nurses were resurveyed, and new inpatient cohorts were assessed. Comparison of preintervention and postintervention time data allowed calculation of provider time reduction, a surrogate measure of improved work efficiency. Prior to the annual consent, >33 hours were spent over 6 months obtaining consent on 40 patients, with >19 hours spent obtaining consent when no transfusions were administered during admission. Twelve months after annual consent implementation, 97.5% (39/40) of analysed patients had a completed annual blood products transfusion consent and provider work efficiency had improved by 94.6% (>30 hours). Although several surveyed variables improved following annual consent implementation, provider frustration with consent process remained 6 out of a max score of 10, the same level as prior to the intervention. Development of an annual inpatient blood products consent form decreased provider time from 15 to <1 min per admission, decreased consenting numbers and increased work efficiency by >90%.

Bleeding Associated with Thrombocytopenia

Platelets are important in the initial formation of the hemostatic plug. Low platelets, or thrombocytopenia, can be caused by multiple abnormalities, ultimately resulting in mucosal and skin bleeding. Immune thrombocytopenia (ITP), heparin-induced thrombocytopenia/thrombosis (HIT), and thrombotic thrombocytopenic purpura (TTP) are three disorders that result from predominantly antibody-mediated platelet destruction and thrombocytopenia. ITP is an acquired autoimmune disorder with multifactorial etiology, including generation of antiplatelet autoantibodies. Treatment in children most often involves observation, but the severity in adults more often requires immunosuppression. HIT is a rare but potentially life-threatening clinical syndrome caused by immune reaction and antibody formation upon heparin exposure. Treatment involves removal of heparin and initiation of direct thrombin or factor Xa inhibitors. TTP is a severe disorder characterized by thrombocytopenia, anemia, and microangiopathic hemolytic anemia secondary to a decrease or absence in the von Willebrand factor protease ADAMTS13. Early recognition and treatment with therapeutic plasma exchange is critical in preventing the morbidity and mortality of the disorder. Platelet refractoriness is the failure to achieve the expected response to a platelet transfusion secondary to both non-immune and immune etiologies. Treatment involves transfusion of compatible platelets and treatment of an underlying triggering condition. The platelet function disorders are a group of hereditary or acquired disorders in which platelet function is defective. Treatment involves supportive therapy and platelet transfusion for significant bleeding.