Histone H4-Induced Thrombin Generation in Fresh and "Reconstituted" Blood

Background. Histones are components of nucleosomes and can be released from activated immune cells as well as from necrotic and apoptotic cells. The release of histones is associated with an increased prothrombotic activity. It has been suggested that this activity is caused by the stimulation of platelets, interference with the protein C pathway, changes in red blood cell (RBC) function, etc.  Studies published so far were done in plasma or using purified cells and proteins. We extended these studies by evaluating thrombin (FIIa) generation in fresh and “reconstituted” blood triggered with histone H4 and examined contribution of several proteins on this process.

Methods. Blood from 8 healthy donors (4 male and 4 female, none of them on oral contraceptives; one male and one female donor were drawn twice) was drawn into 3.2% sodium citrate, 0.1 mg/mL corn trypsin inhibitor (CTI; prevents contact pathway activation) and 20 µg/mL H4 were added followed by recalcification. FIIa generation (TG) was monitored in a fluorogenic assay derived from the CAT method. When desired, either monoclonal inhibitory antibodies to factor (F)XIa, FIXa and tissue factor (TF) (αFXIa-2, αFIX-91 and αTF-5, respectively; all at 0.1 mg/mL) or kallikrein inhibitor aprotinin (10 µM) were added to blood. In parallel, TG in washed RBC reconstituted (50/50) either with platelet poor (PPP) or platelet rich (PRP) plasma (“reconstituted blood”) was analyzed at the same conditions. FIIa generation in an autologous plasma was also analyzed.

Results. A. Whole blood.In the absence of any exogenous activator, the lag phase and peak value of TG in CTI blood varied in a wide range (see the table). An addition of H4 to the blood shortened the lag phase and increased peak FIIa concentration for all donors. An addition of αFXIa-2 slightly prolonged the lag phase and slightly suppressed other parameters of FIIa generation. Aprotinin had a somewhat more pronounced effect on the lag phase but only marginally affected other parameters of FIIa generation. αFIX-91 almost completely suppressed H4-initiated FIIa generation, whereas αTF-5 had almost no effect on the process.

Conditions	Lag (min)		Max FIIa (nM)		Max Rate (nM/min)		AUC (nM*min)
	Range	Average ± SD	Range	Average ± SD	Range	Average ± SD	Range	Average ± SD
CTI (n=10)	9 - 23	13.4 ± 4.2	36.6 - 109.5	63.7 ± 25.1	14.5 - 92.6	48.9 ± 26.6	309.3 - 687.8	487.6 ± 130.1
CTI + H4 (n=10)	2.6 - 7.4	4.7 ± 1.4	66.3 - 134.7	99.4 ± 22.4	36.8 - 83.3	57.9 ± 15.4	417.5 - 865.8	571.1 ± 145
CTI + H4 + αFXIa-2 (n=9)	2.4 - 8.5	5.2 ± 1.9	56.6 - 103.9	83.6 ± 13.2	22.9 - 69. 4	44.9 ± 13.2	409.9 - 680.3	522 ± 90.4
CTI + H4 + aprotinin (n=9)	3.3 - 8.1	5.5 ± 1.6	62.3 - 144.2	95.1 ± 25.7	36 - 94.4	58.1 ± 19.4	409.2 - 1056.2	599.5 ± 203.6
CTI + H4 + αFIXa-91 (n=6)	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ
CTI + H4 + αTF-2 (n=6)	3.2 - 7.4	4.9 ± 1.7	65.1 - 128.4	90.1 ± 21.8	43.4 - 169	73.9 ± 47.6	434.1 - 867.7	543.1 ± 166

                                                                 *NQ = Not Quantifiable (in 4 of 6 experiments no FIIa generation was observed and in 2 experiments it was negligible).

B. RBC. In the absence of any exogenous activator, no FIIa generation was observed in 50 min. in RBC reconstituted with PPP. An addition of H4 led to FIIa generation after a lag phase of 17.7±4.3 min with the maximum active FIIa concentration (66.5±12.4 nM) lower than that observed in whole blood. Additions of either αFXIa-2 or aprotinin had no pronounced effect on FIIa generation, whereas no FIIa was observed in the presence of αFIX-91. When RBC were reconstituted with PRP, H4-induced FIIa generation parameters became similar to those observed in whole blood.

C. Autologous PPP. No FIIa generation was observed in CTI PPP in the absence of any exogenous activator. An addition of H4 led to a delayed (25-30 min lag phase) and inefficient FIIa generation (maximum 10-15 nM). αFXIa-2 had no effect on FIIa generation, αFIX-91 completely suppressed it and aprotinin had an intermediate effect.

Conclusions. 1). H4 has a pronounced effect in enhancing FIIa generation in fresh blood and washed RBC reconstituted with PPP and PRP; 2). Inhibition of contact pathway proteins has minor effect on H4-induced FIIa generation; 3). Inhibition of FIXa almost completely abolishes H4-induced FIIa generation; 4). RBC reconstituted with PPP can support H4-induced FIIa generation and platelets enhance this process; 5). H4-induced FIIa generation in PPP is negligible.