Immune response to hemostatic proteins

Hemophilia

Hemophilia is an X-linked bleeding disorder that is caused by a deficiency of factor VIII (hemophilia A) or factor IX (hemophilia B). Coagulation factor replacement therapy of hemophilia may be complicated by the formation of inhibitory or neutralizing antibodies (inhibitors). This side-effect occurs in approximately 25% of the patients with severe hemophilia A, and in about 5% of the patients with mild hemophilia A.

Increased risk of inhibitor formation in mild hemophilia A has been linked to FVIII missense mutations which include R593C in the A2 domain of FVIII. We have analyzed FVIII-specific T cell responses in two unrelated patients heterozygous for HLA-DRB1*1101 using DR1101-HLA class II tetramers. CD4+ T cells reactive with a peptide including the R593 that was modified in the FVIII protein of both patients were observed (see figure). Our findings support the concept that presentation of peptides specifically derived from wild type and not endogenously expressed factor VIII promote the formation of inhibitors in patients with mild hemophilia A carrying the appropriate HLA-class II alleles. These findings contribute to our knowledge as to why inhibitors develop in a subset of patients with mild hemophilia A.

PP HLA-DR11 tetramer guided selection of FVIII-specific CD4+ T cells
HLA-DR11 tetramer guided selection of FVIII-specific CD4+ T cells in a patient with mild hemophilia A. Arrow indicates the population of cells that is stained by HLA-DR11 tetramers loaded with FVIII peptide containing R593.

Thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a micro-angiopathy that is related to an acquired or congenital deficiency of the von Willebrand Factor (VWF) cleaving protease ADAMTS13. In the absence of ADAMTS13, ultra large VWF (UL-VWF) polymers, originating from endothelial cell specific organelles, designated Weibel-Palade bodies, accumulate in the circulation. These UL-VWF polymers mediate the formation of platelet-rich thrombi in the microcirculation that give rise to hemolytic anemia and thrombocytopenia. In plasma of the majority of patients with acquired TTP, antibodies directed towards ADAMTS13 are present. The majority of patients develop antibodies directed towards the spacer domain of ADAMTS13. In a recent study we have shown that in exposed surface in the spacer domain comprising residues R660, Y661 and Y665 contribute to the binding of anti-ADAMTS13 antibodies (see figure). Replacement of these residues also affected the ability of ADAMTS13 to process small VWF substrates. Also the processing of full length VWF under denaturing conditions or under flow on the surface of endothelial cells was greatly impaired by replacement of R660, Y661 and Y665 by an alanine. Using complementary VWF variants we were able to show that the exposed surface formed by R660, Y661 and Y665 interacts with a conserved alpha-helix in the A2 domain of VWF. This part of the A2 domain unfolds under the influence of shear stress allowing it to interact with ADAMTS13. Our findings suggest that human antibodies directed towards residues R660, Y661 and Y665 in the spacer domain interfere with productive assembly of the VWF-ADAMTS13 complex thereby interfering with cleavage of ultra large VWF multimers on the surface of endothelial cells.

PP exposed surface containing residues R660, Y661 and Y665
Exposed surface containing residues R660, Y661 and Y665 (indicated in blue) in the spacer domain (indicated in green) of ADAMTS13 that is crucially involved in the binding of anti-ADAMTS13 antibodies. Water molecules are displayed in orange.

Key publications